KR20140107658A - Mglu 2/3 agonists - Google Patents

Abstract

The present invention provides novel mGlu2 / 3 agonists of formula (I) useful for the treatment of neurological or psychiatric disorders.
(I)

Description

MGLU 2/3 Agents {MGLU 2/3 AGONISTS}

The present invention relates to mGlu2 / 3 agonists, more particularly novel 4-substituted bicyclo [3.1.0] hexane and its prodrugs, pharmaceutical compositions thereof and therapeutic uses thereof.

L-glutamate is a major excitatory neurotransmitter in the central nervous system and is referred to as an excitatory amino acid. Metabolic glutamate (mGlu) receptors are G-protein-coupled receptors that regulate neuronal excitability. Treatment of neurological or psychiatric disorders has been linked to the selective activation of mGlu excitatory amino acid receptors. More particularly, the study demonstrates that mGlu2 / 3 agonists have analgesic, antipsychotic, anxiolytic, antidepressant and neuroprotective properties. Thus, this property of the mGlu2 / 3 agonist may be useful in the treatment of neurological disorders such as chronic pain states or psychiatric disorders such as schizophrenia, bipolar disorder (also known as jaw disorder), generalized anxiety disorder and post-traumatic stress disorder . ≪ / RTI >

WO 9717952 discloses certain 4-substituted bicyclo [3.1.0] hexane compounds claimed to be excitatory amino acid receptor antagonists.

Excess glutamatergic tensions are involved in many disease states of the central nervous system; Effective agents for correcting these pathophysiological conditions are lacking in clinical practice. In particular, clinical applications have not been realized due to the lack of mGlu2 / 3 agonists with appropriate drug-like properties. Therefore, there is still a need for a potent and effective mGlu2 / 3 agonist. The present invention provides novel 4-substituted bicyclo [3.1.0] hexane and its prodrugs which are potent and effective mGlu2 / 3 agonists. Certain prodrugs within the scope of this invention are well absorbed after oral administration and subsequently hydrolyzed to release active metabolites into the systemic circulation and are therefore suitable for clinical development. Such novel compounds of the present invention are useful for the treatment of neurological disorders such as chronic pain states such as persistent pain, neuropathic pain, chronic inflammatory pain or visceral pain, or psychiatric disorders such as schizophrenia, bipolar disorder, generalized anxiety disorder or post- The need for a powerful and effective treatment of stress disorders was addressed.

The present invention provides a compound of formula (I), or a pharmaceutically acceptable salt thereof.

(I)

here,

R ¹ is hydrogen, R ² is hydrogen, and R ³ is hydrogen;

R ¹ is hydrogen, R ² is (2S) -2-aminopropanoyl, and R ³ is hydrogen;

R ¹ is hydrogen, R ² is (2S) -2-amino-4-methylsulfanyl-butanoyl and R ³ is hydrogen;

R ¹ is hydrogen, R ² is 2-aminoacetyl, and R ³ is hydrogen;

R ¹ is benzyl, R ² is hydrogen and R ³ is benzyl; or

R ¹ is (2-fluorophenyl) methyl, R ² is hydrogen, and R ³ is (2-fluorophenyl) methyl.

The present invention relates to the use of (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6- dicarboxylic acid or a pharmaceutically acceptable salt thereof Salt.

In a particular embodiment, the present invention provides a process for the preparation of (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6- dicarboxylic acid to provide.

In a particular embodiment, the present invention relates to (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid Hydrochloride salt.

The present invention relates to (1S, 2S, 5R, 6S) -2 - [[(2S) -2-aminopropanoyl] amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] 2,6-dicarboxylic acid or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the present invention relates to the use of (1S, 2S, 5R, 6S) -2 - [[(2S) -2-aminopropanoyl] amino] spiro [bicyclo [3.1.0] hexane- -Cyclopropane] -2,6-dicarboxylic acid hydrochloride.

In a particular embodiment, the present invention relates to the use of (1S, 2S, 5R, 6S) -2 - [[(2S) -2-aminopropanoyl] amino] spiro [bicyclo [3.1.0] hexane- - cyclopropane] -2,6-dicarboxylic acid; 1,4-dioxane (1: 0.5); Hydrochloride.

The present invention relates to a process for the preparation of (1S, 2S, 5R, 6S) -2 - [[(2S) -2-amino-4-methylsulfanyl- butanoyl] amino] spiro [bicyclo [3.1.0] hexane- -Cyclopropane] -2,6-dicarboxylic acid or a pharmaceutically acceptable salt thereof.

(2S) -2-amino-4-methylsulfanyl-butanoyl] amino] spiro [bicyclo [3.1.0] < RTI ID = 0.0 > Hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid hydrochloride.

The present invention relates to a process for the preparation of (1S, 2S, 5R, 6S) -2 - [(2-aminoacetyl) amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] 0.0 > pharmaceutically < / RTI > acceptable salts thereof.

In a particular embodiment, the present invention provides a pharmaceutical composition comprising (1S, 2S, 5R, 6S) -2 - [(2-aminoacetyl) amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] , 6-dicarboxylic acid hydrochloride.

The present invention relates to a process for the preparation of dibenzyl (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6- Provide acceptable salts.

In a particular embodiment, the present invention provides a process for the preparation of dibenzyl (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6- Rate; 1,4-dioxane; Hydrochloride.

The present invention relates to bis [(2-fluorophenyl) methyl] (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -Dicarboxylate or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the present invention relates to a process for the preparation of bis [(2-fluorophenyl) methyl] (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane ] -2,6-dicarboxylate hydrochloride.

The present invention relates to the use of (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6- dicarboxylic acid or a pharmaceutically acceptable salt thereof [(2S) -2-aminopropanoyl] amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2 , 6-dicarboxylic acid or a pharmaceutically acceptable salt thereof, (1S, 2S, 5R, 6S) -2 - [[(2S) (1S, 2S, 5R, 6S) -2 - [(2- (4-methylpiperazin-1-yl) (1S, 2S, 5R, 2S, 5R, 6R) aminoacetyl) amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid or a pharmaceutically acceptable salt thereof, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate or a pharmaceutically acceptable salt thereof, or bis [ Phenyl) methyl] (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane- 4,1'-cyclopropane] -2,6-dicarboxylate or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier and optionally other therapeutic ingredients.

The present invention relates to the use of (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6- dicarboxylic acid or a pharmaceutically acceptable salt thereof [(2S) -2-aminopropanoyl] amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2 , 6-dicarboxylic acid or a pharmaceutically acceptable salt thereof, (1S, 2S, 5R, 6S) -2 - [[(2S) (1S, 2S, 5R, 6S) -2 - [(2- (4-methylpiperazin-1-yl) (1S, 2S, 5R, 2S, 5R, 6R) aminoacetyl) amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid or a pharmaceutically acceptable salt thereof, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate or a pharmaceutically acceptable salt thereof, or bis [ Phenyl) methyl] (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane- 4,1'-cyclopropane] -2,6-dicarboxylate or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

(1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] - 2,6-dicarboxylic acid or a pharmaceutically acceptable salt thereof, (1S, 2S, 5R, 6S) -2 - [[(2S) -2-aminopropanoyl] amino] spiro [bicyclo [3.1. (1S, 2S, 5R, 6S) -2 - [[(2S) -2-amino-1-methylpropanoic acid & -4-methylsulfanyl-butanoyl] amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid or a pharmaceutically acceptable salt thereof, Amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid or its pharmaceutical (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate or a salt thereof A pharmaceutically acceptable salt, or a bis [(2- (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate or its pharmaceutical Which method comprises administering a therapeutically effective amount of a pharmaceutically acceptable salt.

(1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] - 2,6-dicarboxylic acid or a pharmaceutically acceptable salt thereof, (1S, 2S, 5R, 6S) -2 - [[(2S) -2-aminopropanoyl] amino] spiro [bicyclo [3.1. (1S, 2S, 5R, 6S) -2 - [[(2S) -2-amino-1-methylpropanoic acid & -4-methylsulfanyl-butanoyl] amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid or a pharmaceutically acceptable salt thereof, Amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid or its pharmaceutical (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate or a salt thereof A pharmaceutically acceptable salt, or a bis [(2-fluoro (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate or its pharmaceutical Provides the use of acceptable salts.

The present invention relates to the use of (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid Or a pharmaceutically acceptable salt thereof; (1S, 2S, 5R, 6S) -2 - [[(2S) -2-aminopropanoyl] amino] spiro [bicyclo [3.1.0] hexane- - cyclopropane] -2,6-dicarboxylic acid or a pharmaceutically acceptable salt thereof, (1S, 2S, 5R, 6S) -2 - [[(2S) (1S, 2S, 5R, 6S) - cyclohexyl] -2,6-dicarboxylic acid or a pharmaceutically acceptable salt thereof, Bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid or a pharmaceutically acceptable salt thereof, dibenzyl (2-aminoethyl) 1, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate or a pharmaceutically acceptable salt thereof, [(2-fluorophenyl) methyl] (1S, 2S, 5R, 6S) -2-aminos To [bicyclo [3.1.0] hexane -4,1'- cyclopropane] provides a 2,6-dicarboxylate or a pharmaceutically acceptable salt thereof. The present invention also relates to the use of (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] 6-dicarboxylic acid or a pharmaceutically acceptable salt thereof, (1S, 2S, 5R, 6S) -2 - [[(2S) -2-aminopropanoyl] amino] spiro [bicyclo [ (1S, 2S, 5R, 6S) -2 - [[(2S) -2-amino-4-methylpropanoic acid or its pharmaceutically acceptable salt, Cyclopropyl] -2,6-dicarboxylic acid or a pharmaceutically acceptable salt thereof, (1S, 2S (2S, 3S) , 5R, 6S) -2 - [(2-aminoacetyl) amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6- dicarboxylic acid, (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate or a pharmaceutical salt thereof Acceptable salts, or bis [(2-fluorophenyl ) Methyl] (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6- dicarboxylate or a pharmaceutically acceptable salt thereof Provide a salt

In addition, the present invention provides a method of treating a condition selected from the group consisting of persistent pain, neuropathic pain, chronic inflammatory pain and visceral pain, wherein the mental disorder is selected from the group consisting of schizophrenia, bipolar disorder, generalized anxiety disorder, , Preferred embodiments of methods and uses as described herein.

Unless indicated otherwise, the following terms, as used above and throughout the description of the present invention, shall be understood to have the following meanings:

"Compound (s) of the present invention" includes prodrugs, active compounds and / or active metabolites. A "prodrug" is a class of drugs that are initially in an inactive form that is converted into the active form in the body by normal metabolic processes, wherein at least one of R ¹ , R ^2, and R ³ in Formula I is other than hydrogen For example, R ¹ is hydrogen, R ² is (2S) -2-aminopropanoyl, R ³ is hydrogen, R ¹ is hydrogen and R ² is (2S) methylsulfanyl-butanoyl, and, R ³ is hydrogen; and R ¹ is hydrogen, R ² is a 2-amino-acetyl, R ³ is hydrogen; and R ¹ is benzyl, R ² is hydrogen, R ³ is Benzyl, or R ¹ is (2-fluorophenyl) methyl, R ² is hydrogen and R ³ is (2-fluorophenyl) methyl. &Quot; Active compound "or" Active "is another designation for an active form that is administered into the body, for example intravenously or intraperitoneally, wherein R ¹ , R ² and R ³ are hydrogen. "Active metabolite" is another designation for the active form resulting from the conversion of prodrugs in the body by normal metabolic processes, wherein R ¹ , R ² , and R ³ are hydrogen.

"Pharmaceutically acceptable carrier, diluent or excipient" is a medium generally accepted in the art for delivering biologically active agents to mammals, such as humans.

"Pharmaceutically acceptable salts" refer to the relatively non-toxic inorganic and organic salts of the compounds of the present invention.

A "therapeutically effective amount" or "effective amount" is intended to encompass any of the inventive compounds, compositions, compositions, pharmaceutical compositions, compositions, ≪ / RTI > or a pharmaceutically acceptable salt thereof, or a compound of the invention, or a pharmaceutical acceptable salt thereof.

The terms " treatment, "" treating," " treating, " and the like, include slowing or reversing the progression of the disorder. These terms are also intended to mean alleviating, alleviating, abating, reducing or reducing one or more symptoms of a disorder or condition, even if the disorder or condition is not actually eliminated, and even if the progress of the disorder or condition itself is delayed or not reversed .

Under standard nomenclature used throughout this disclosure, the terminal portion of the specified side chain is first described, followed by the functional group adjacent to the point of attachment. For example, the methylsulfonyl substituent is equivalent to CH ₃ -SO ₂ -.

The compounds of the invention may, for example, react with a number of inorganic and organic acids to form pharmaceutically acceptable acid addition salts or base addition salts. Such pharmaceutically acceptable salts and conventional methodology for preparing them are well known in the art. See, e.g., P. Stahl, et al., HANDBOOK OF PHARMACEUTICAL SALTS: PROPERTIES, SELECTION AND USE, (VCHA / Wiley-VCH, 2002); S.M. Berge, et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, Vol. 1, January 1977].

The compounds of the present invention are preferably formulated as a pharmaceutical composition using a pharmaceutically acceptable carrier, diluent or excipient and administered by various routes. Preferably, such compositions are for oral or intravenous administration. Such pharmaceutical compositions and methods for making them are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy (A. Gennaro, et al., Eds., 21st ed., Mack Publishing Co., 2005).

The compound of the present invention to be actually administered will be determined by the physician under the relevant circumstances including the condition to be treated, the route of administration chosen, the actual compound or compounds of the present invention administered, the age, weight and response of the individual patient, and the severity of the patient's symptoms. The daily dose is usually in the range of about 0.1 to about 300 mg. In some cases dosage levels below the lower limit of the above-mentioned range may be much more sufficient, but in other cases much larger doses may be used.

The compounds of the present invention or pharmaceutically acceptable salts thereof may be prepared by various procedures known in the art as well as the procedures described in the following Preparation Examples and Examples. Specific synthetic steps for each route described can be combined in various ways to produce the compounds of the present invention, or pharmaceutically acceptable salts thereof.

Unless otherwise indicated, the substituents are as previously defined. Reagents and starting materials are generally readily available to those skilled in the art. Others may be prepared by the procedures described in the following preparations and examples, including the standard techniques of organic and heterocyclic chemistry, techniques similar to the synthesis of known structurally similar active compounds and prodrugs, and any novel procedures . The naming of the following preparations and examples is carried out using Symyx Draw 3.1.

The following terms used herein have the indicated meanings: "HPLC" refers to high pressure liquid chromatography; "LC" refers to liquid chromatography; "MS (ES +)" refers to mass spectrometry using electrospray ionization; "MS" refers to mass spectrometry; "SFC" refers to supercritical fluid chromatography; "NMR" refers to nuclear magnetic resonance; "TLC" refers to thin layer chromatography; "RT" refers to the residence time; "UV" refers to ultraviolet light; "EDTA" refers to ethylenediamine tetraacetic acid; "PBS" refers to phosphate buffered saline; "PCR" refers to polymerase chain reaction; "SCX" refers to strong cation exchange; "HLB" refers to hydrophilic-lipophilic balance.

Production Example 1

(1S, 2S, 5R, 6S) -2- (tert-butoxycarbonylamino) -4-methylene-bicyclo [3.1.0] hexane-2,6-dicarboxylate

The oven-dried 250 mL round bottom flask was charged with methyltriphenylphosphonium bromide (5.41 g, 14.9 mmol) and tetrahydrofuran (93 mL). The suspension was cooled to 0 C and a 1M solution of sodium bis (trimethylsilyl) amide (16.08 mL, 16.08 mmol) in tetrahydrofuran was added dropwise. After stirring the pale yellow mixture at 0 C for 20 min, di-tert-butyl (1S, 2S, 5R, 6R) -2- (tert- butoxycarbonylamino) -4 (5.09 g, 12.4 mmol, see WO03 / 104217 / A2 for synthesis details) was added to a solution of 3-bromo-3-oxo-bicyclo [3.1.0] hexane-2,6-dicarboxylate. The reaction was allowed to warm to room temperature and stirred for 16 h. The mixture was partitioned between ethyl acetate (500 mL) and water (350 mL). The aqueous phase was discarded and the organic phase was washed with brine (200 mL). The organic phase was dried over magnesium sulfate, filtered and concentrated under reduced pressure. Purification by flash chromatography eluting with ethyl acetate: iso-hexane (5:95 to 20:80) afforded the title compound (4.84 g, 11.2 mmol) as a white solid.

Production Example 2

(1S, 2S, 5R, 6S) -2- (tert-butoxycarbonylamino) spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6- Leucylate

An ethanol-free solution of diazomethane in diethyl ether was prepared by filling the cold-fingers of an Aldrich mini-Diazald apparatus with card ice and iso-propanol and filling it 1/3. To a portion of the reaction of the apparatus was placed a solution of potassium hydroxide (740 mg, 13.2 mmol) in water (1.5 mL). To this solution was added a mixture of diethylene glycol monoethyl ether (4 mL) and diethyl ether (2.4 mL). N-methyl-N'-nitro-N-nitrosoguanidine (1 g, 6.8 mmol) was dissolved in a mixture of diethyl ether (13 mL) and diethylene glycol monoethyl ether (4 mL). The solution was placed in a dropping funnel with smooth glass connections and mounted in a mini-diazal device. The potassium hydroxide solution was warmed in the water bath maintained at 70 DEG C while confirming that both the collection flask filled with diethyl ether at the outlet of the apparatus and the bubbler were cooled in the cardiis / iso-propanol bath. A solution of N-methyl-N'-nitro-N-nitrosoguanidine was added at the same rate as the distillate produced. Once the distillation was complete, additional diethyl ether was added dropwise via a dropping funnel until the condensate on the cold finger became colorless. The resulting solution was stored in a card ice / iso-propanol bath until needed. Palladium acetate (17.2 mg; 76.5 [mu] mol) in diethyl ether (10 mL) was suspended. The resulting light brown solution was triturated and filtered to give the diazomethane solution di tert-butyl (1S, 2S, 5R, 6S) -2- (tert- butoxycarbonylamino) -4-methylene-bicyclo [3.1. 0] hexane-2,6-dicarboxylate (330 mg, 765.5 [mu] mol) and diethyl ether (10 mL) and allowed to reach ambient temperature. Once the gas evolution ceased, the resulting suspension was filtered and concentrated under reduced pressure to give the unreacted ditert-butyl (1S, 2S, 5R, 6S) -2- (tert- butoxycarbonylamino) Bicyclo [3.1.0] hexane-2,6-dicarboxylate and 260 mg of the title compound was obtained.

Potassium hydroxide (2.5 g, 44.6 mmol), and diethyl ether (30 mL) and diethyleneglycol monoethyl ether in diethyleneglycol monoethyl ether (14 mL) and water (4 mL) An ethanol-free solution of diazomethane in diethyl ether from a solution of N-methyl-N'-nitro-N-nitrosoguanidine (4 g, 27.2 mmol) in a mixture of tetrahydrofuran (15 mL) A solution of the resulting diazomethane was taken and this was added to a solution of unreacted ditert-butyl (1S, 2S, 5R, 6S) -2- (tert-butoxycarbonylamino) -Bicyclo [3.1.0] hexane-2,6-dicarboxylate and the title compound (260 mg) in 30 ml of a suspension of palladium acetate (20 mg, 89.1 [ Was added little by little. Once gas evolution ceased, it was allowed to stand overnight, then filtered through a phase separator frit and concentrated under reduced pressure to give a dark oil. Purification by flash chromatography eluting with ethyl acetate: iso-hexane (0: 100 to 75:25) gave the unreacted ditert-butyl (1S, 2S, 5R, 6S) -2- (tert- butoxycarbonylamino ) -4-methylene-bicyclo [3.1.0] hexane-2,6-dicarboxylate and 185 mg of the title compound.

Potassium hydroxide (3.2 g, 57 mmol) and diethyl ether (35 mL) in diethyleneglycol monoethyl ether (4 mL) and diethyleneglycol monoethyl ether (10 mL) in water (5 mL) 15 mL) was prepared an ethanol-free solution of diazomethane in diethyl ether from a solution of N-methyl-N'-nitro-N-nitrosoguanidine (5 g, 34 mmol). A solution of the resulting diazomethane was taken and this was added to a solution of unreacted ditert-butyl (1S, 2S, 5R, 6S) -2- (tert-butoxycarbonylamino) -Bicyclo [3.1.0] hexane-2,6-dicarboxylate and the title compound (185 mg) in a mixture of palladium acetate (20 mg, 89.1 [ Was added little by little. Once gas evolution ceased, it was allowed to settle for 30 minutes, filtered and concentrated under reduced pressure to give a yellow oil. Water-w / 0.1% formic acid; Gradient: 28-62% in 1.35 min < tb >< tb > ______________________________________ < tb > ______________________________________ < Purification by acetonitrile w / 0.1% formic acid followed by 62-95% in 6.65 min followed by 95% over 3.55 min. Column temperature: ambient; flow rate: 45 mL / min) afforded the title compound as a colorless oil , 130.6 [mu] mol).

Production Example 3

Diethyl (1S, 2S, 5R, 6R) -2-amino-4-oxo-bicyclo [3.1.0] hexane-2,6-dicarboxylate.

A 500 mL three-necked flask equipped with a condenser, nitrogen inlet and thermometer was added to a solution of ditert-butyl (1S, 2S, 5R, 6R) -2- (tert- butoxycarbonylamino) -Bicyclo [3.1.0] hexane-2,6-dicarboxylate (15 g, 36.4 mmol). Thionyl chloride (13.3 mL, 182.3 mmol) was added via syringe to the stirred solution at room temperature (weak exotherm) and then heated to reflux. After 24 hours, the reaction was cooled to room temperature and concentrated in vacuo. The residue in dichloromethane (50 mL) was dissolved and then concentrated on a rotary evaporator (repeated 3 times). The residue was partitioned between ethyl acetate (200 mL) and saturated sodium hydrogenated carbonate (150 mL). The organic phase was washed with brine (150 mL), dried over sodium sulfate, filtered, and then concentrated to dryness to give the title compound (8.24 g, 32.3 mmol) as an oil.

Production Example 4

Diethyl (1S, 2S, 5R, 6R) -2-acetamido-4-oxo-bicyclo [3.1.0] hexane-2,6-dicarboxylate.

Acetic anhydride (5 mL, 50.8 mmol) was added to a solution of diethyl (1S, 2S, 5R, 6R) -2-amino-4-oxo-bicyclo [3.1.0] hexane- -Dicarboxylate (7.2 g, 28.2 mmol) and triethylamine (7.9 mL, 56.4 mmol) at room temperature. After 2 hours, quenching with water (100 mL) and vigorous stirring for 20 minutes. The dichloromethane layer was separated via a hydrophobic frit and evaporated to a pale brown oil (9.6 g). Purification by flash chromatography eluting with ethyl acetate: iso-hexane (70:30 → 90:10) afforded the title compound (6.53 g, 22 mmol) as a pale yellowish foam which foamed on drying.

Production Example 5

Diethyl (1S, 2S, 5R, 6S) -2-acetamido-4-methylene-bicyclo [3.1.0] hexane-2,6-dicarboxylate.

The oven-dried 250 mL round bottom flask was charged with (methyl) triphenylphosphonium bromide (9.72 g, 26.7 mmol) and dry tetrahydrofuran (122 mL). The suspension was cooled to 0-5 < 0 > C and 2M sodium bis (trimethylsilyl) amide in tetrahydrofuran (13.3 mL, 26.7 mmol) was treated dropwise. The resulting pale yellow mixture was then stirred at 0 < 0 > C for 20 min, and diethyl (1S, 2S, 5R, 6R) -2- acetamido-4-oxo-bicyclo [3.1 0] hexane-2,6-dicarboxylate (6.1 g, 20.5 mmol) in dichloromethane. The reaction was allowed to slowly warm to room temperature over 3 hours. After stirring at room temperature for 20 hours, quenched with cold water (200 mL) and extracted with ethyl acetate (200 mL). The extract was washed with water (100 mL), brine (100 mL), dried, filtered and evaporated to a dark brown oil. Diethyl ether (70 mL) and iso-hexane (20 mL) were added and the solution was seeded with triphenylphosphine oxyd. Allowed to settle for 2 hours, the solution was decanted, silica was added and concentrated in vacuo. Purified by flash chromatography eluting with ethyl acetate: iso-hexane (60:40 - > 80:20) to give a pink oil (6.81 g) contaminated with triphenylphosphine oxide. The title compound (3.98 g, 13.5 mmol) was obtained as a viscous yellow oil by flash chromatography eluting with ethyl acetate: iso-hexane (60:40).

Production Example 6

Diethyl (1S, 2S, 5R, 6S) -2-acetamidospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate

A solution of trifluoroacetic acid (1.9 mL, 25 mmol) in dichloromethane (12.5 mL) under nitrogen was added to a cold (ice bath) solution of diethylzinc (1 M in heptane) (25 mL, 25 mmol) in dichloromethane ) Was slowly added dropwise to the stirring solution. After 10 minutes, a solution of diiodomethane (2.01 mL, 25 mmol) in dichloromethane (12.5 mL) was added. After 10 minutes, a solution of diethyl (1S, 2S, 5R, 6S) -2-acetamido-4-methylene- bicyclo [3.1.0] hexane- 2,6-dicarboxylate in dichloromethane (12.5 mL) (2.46 g, 8.3 mmol) in dichloromethane was added. After 60 minutes the ice bath was removed and the reaction mixture was allowed to stir at room temperature overnight. Under vigorous stirring, the clear reaction solution was quenched by the dropwise addition of the reaction mixture with 0.2 M aqueous hydrochloric acid (200 mL). After 1 h, the dichloromethane layer was separated, washed with brine, dried over magnesium sulfate, filtered and concentrated in vacuo to give the required product (4.4 g) contaminated with unreacted starting material. Purification by flash chromatography eluting with ethyl acetate: cyclohexane (50:50 to 100: 0) gave a colorless oil (3.1 g). SFC (RT = 2.48 min (UV, 200 nm); HPLC column: AD-H 30 mm x 250 mm 5 μm; CO ₂ gradient: 5% iso-propyl alcohol w / 0.2% dimethylethylamine followed by 2.2 (1S, 2S, 5R, 6S) -2-acetamido-benzoic acid as a colorless oil, 4-methylene-bicyclo [3.1.0] hexane-2,6-dicarboxylate (580 mg, 2.0 mmol) and the title compound (1.82 g, 5.9 mmol).

Production Example 7

Diethyl (1S, 2S, 5R, 6S) -2-amino-4-methylene-bicyclo [3.1.0] hexane-2,6-dicarboxylate.

Method 1:

A solution of approximately 1 M hydrogen chloride in ethanol was prepared by dropwise adding trimethylsilyl chloride (13.8 mL, 108 mmol) to ethanol (110 mL). To a solution of ditert-butyl (1S, 2S, 5R, 6S) -2- (tert-butoxycarbonylamino) -4-methylene-bicyclo [3.1.0] hexane- (7.4 g, 18.1 mmol) was dissolved and heated to 60 < 0 > C for 16 h. Evaporated to a viscous yellow oil, redissolved in water (50 mL), and the cloudy solution was filtered to remove traces of insoluble matter. The aqueous acidic solution was neutralized with sodium bicarbonate (8.4 g, 0.1 mol) and extracted with dichloromethane (2 x 100 mL). The combined dichloromethane solution was dried over a hydrophobic frit and evaporated to a pale yellow liquid (4.14 g). Purification by chromatography on amino-bonded silica eluting with ethyl acetate: iso-hexane (20:80 - > 80:20) gave a colorless liquid (3.6 g). Purification by chromatography on amino-bonded silica eluting with ethyl acetate: iso-hexane (20:80) gave a colorless liquid of the title product (2.81 g, 61%).

Method 2:

p-Toluenesulfonic acid monohydrate (6.97 g, 36.6 mmol) was dried under vacuum at 50 < 0 > C for 3 days. (1S, 2S, 5R, 6S) -2- (tert-butoxycarbonylamino) -4-methylene-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid (5.0 g, 12.2 mmol) in tetrahydrofuran and heated to 60 < 0 > C for 3 days. The solvent was removed under reduced pressure to give a residue. The residue was dissolved in water (100 mL), made basic with sodium bicarbonate, and extracted with dichloromethane (3 x 100 mL). The extract was dried over sodium sulfate, filtered and then concentrated under reduced pressure to give an orange residue of the title compound (1.52 g, 51%).

Production Example 8

Diethyl (1S, 2S, 5R, 6S) -2- (9H-fluoren-9-ylmethoxycarbonylamino) -4-methylene- bicyclo [3.1.0] hexane-2,6-dicarboxylate

(3.15 g, 12.2 mmol) was added dropwise to a solution of diethyl (1S, 2S, 5R, 6S) -2- (4-fluorophenyl) To a stirred solution of 2-amino-4-methylene-bicyclo [3.1.0] hexane-2,6-dicarboxylate (2.8 g, 11.1 mmol) and sodium bicarbonate (2.04 g, 24.3 mmol) Lt; / RTI > After 1 hour, water (10 mL) was added and extracted with ethyl acetate (50 mL). The extract was washed with brine solution (20 mL), dried, filtered and evaporated to a pale yellow oil (6.5 g). Purification by flash chromatography eluting with ethyl acetate: iso-hexane (10:90 to 20:80) gave a viscous colorless oil (4.58 g). Refinement by flash chromatography eluting with ethyl acetate: iso-hexane (20:80) afforded the title compound as a white semi-solid foam (4.22 g, 80%).

Production Example 9

Spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,2-di (1S, 2S, 5R, 6S) -2- 6-dicarboxylate

크로마토그래피를 반복함으로써 보다 덜 순수한 분획으로부터의 생성물의 제2 로트 (363 mg, 추가의 9%)를 수득하였다.A solution of trifluoroacetic acid (2.66 g, 23.3 mmol) in dichloromethane (11.7 mL) was treated with diethylzinc 1 M (23.3 mL, 23.3 mmol) in heptane in dichloromethane (11.7 mL) cooled to 0-5 & Was added dropwise over 5 minutes (exothermic reaction). After 10 minutes, a solution of diiodomethane (6.25 g, 23.3 mmol) in dichloromethane (11.7 mL) was added. After 10 minutes, a solution of diethyl (lS, 2S, 5R, 6S) -2- (9H-fluoren-9-ylmethoxycarbonylamino) -4- methylene- bicyclo [3.1.0 ] Hexane-2,6-dicarboxylate (3.70 g, 7.8 mmol). After 2 hours at 0 C, it was allowed to warm to room temperature and stirred for 16 hours. The reaction mixture was quenched with cold 0.5 M hydrochloric acid (50 mL) and dichloromethane (20 mL) and vigorously stirred. The dichloromethane layer was separated via a hydrophobic frit and then evaporated to a pale yellow oil. Purification by flash chromatography eluting with ethyl acetate: iso-hexane (10:90 to 20:80) yielded a colorless oil (3.49 g). The intermediate fraction was taken up by flash chromatography eluting with ethyl acetate: iso-hexane (20:80) to give a colorless foam of the title compound (2.12 g, 56%).

Chromatography was repeated to give a second lot (363 mg, additional 9%) of the product from the less pure fractions.

Production Example 10

Diethyl (1S, 2S, 5R, 6R) -2-benzyloxycarbonylamino-4-oxo-bicyclo [3.1.0] hexane-2,6-dicarboxylate

Method 1:

Dibenzyl dicarbonate (18.6 mL, 76.1 mmol) was added to a solution of diethyl (1S, 2S, 5R, 6R) -2-amino-4-oxo-bicyclo [3.1.0] hexane- , 6-dicarboxylate (16.2 g, 63.5 mmol) in dichloromethane (10 mL), followed by the dropwise addition of triethylamine (10.6 mL, 76.1 mmol). After washing with 1N hydrochloric acid (200 mL), the reaction mixture was stirred for 30 minutes. The dichloromethane phase was washed with brine, separated and concentrated in vacuo. Purification by flash chromatography eluting with ethyl acetate: iso-hexane (0: 100 to 75:25) gave the title compound (11.6 g, 47%).

Method 2:

(1S, 2S, 5R, 6R) -2-amino-4-oxo-bicyclo [3.1.0] hexane-2,6-dicarboxylate hydrochloride (226.86 g, 777.65 mmol) , Water (1.13 L) and tetrahydrofuran (1.13 L). Sodium bicarbonate (143.72 g, 1.71 mol) was slowly added in five portions (CO ₂ evolution and internal temperature from 31 ° C to 25 ° C observed). A solution of benzyl chloroformate (120.6 mL, 855.42 mmol) in tetrahydrofuran (226.9 mL) was then added, the internal temperature was kept below 28 DEG C (10 min) and the reaction was stirred at 25 DEG C for 1 h Respectively. The mixture was poured into methyl-t-butyl ether (1.25 L). The layers were separated, the aqueous phase was extracted with ethyl acetate (750 ml), and the aqueous phase was discarded. The mixture was washed with brine, dried over magnesium sulfate, filtered and concentrated to dryness to give the title compound (302.82 g, 777.65 mmol) as a colorless oil.

Production Example 11

Diethyl (1S, 2S, 5R, 6S) -2-benzyloxycarbonylamino-4-methylene-bicyclo [3.1.0] hexane- 2,6-dicarboxylate

Method 1:

The oven dried 500 mL round bottom flask was charged with (methyl) triphenylphosphonium bromide (15.4 g, 43.1 mmol) and dry tetrahydrofuran (112 mL). The suspension was cooled to 0-5 < 0 > C and 2M sodium bis (trimethylsilyl) amide (23 mL, 46 mmol) in tetrahydrofuran was treated dropwise. The light yellow suspension produced at 0 C for 30 min was stirred and then a solution of diethyl (1S, 2S, 5R, 6R) -2-benzyloxycarbonylamino-4-oxo-bicyclo [ 3.1.0] hexane-2,6-dicarboxylate (11.2 g, 28.8 mmol). The reaction was allowed to slowly warm to room temperature. After 4 hours, it was quenched with ice (120 g), brine (120 mL) and extracted with ethyl acetate (250 mL). The extract was washed with brine (2 x 100 mL), dried, filtered and evaporated to a red oil. Redissolved in diethyl ether (100 mL), and iso-hexane (80 mL) was added in several portions to slowly add triphenylphosphine oxide (5.2 g) as a red semi-solid. The solution was treated with dry silica (~ 50 g) and concentrated to dryness. Purification by flash chromatography eluting with ethyl acetate: iso-hexane (20:80) afforded the title compound (7.37 g, 66%) as a colorless viscous oil.

Method 2:

Potassium tert-butoxide (104.72 g, 933.18 mmol) was added to a suspension of (methyl) triphenylphosphonium bromide (340.16 g, 933.18 mmol) in tetrahydrofuran (1.82 L) at room temperature without internal temperature change. Then, a solution of diethyl (1S, 2S, 5R, 6R) -2-benzyloxycarbonylamino-4-oxo-bicyclo [3.1.0] hexane- A solution of 2,6-dicarboxylate (302.82 g, 777.65 mmol) was added. The mixture was stirred at 50 < 0 > C for 3 hours. The reaction was diluted with ethyl acetate (1.5 L), washed with water (2 x 3 L) and brine. The organic phase was dried over magnesium sulfate, filtered and concentrated to give a dark brown oil. Purification by flash chromatography eluting with ethyl acetate: hexane (9: 1) gave the title compound (183.35 g, 473.25 mmol) as a colorless oil.

Production Example 12

Diethyl (1S, 2S, 5R, 6S) -2-benzyloxycarbonylaminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate

Method 1:

A solution of 1M diethylzinc (64.1 mL, 64.1 mmol) in heptane in dichloromethane (14.6 mL) was added to a solution of trifluoroacetic acid (4.27 mL, 56.5 mmol) in dichloromethane (73 mL) Lt; / RTI > over 10 minutes. After 10 minutes, a solution of diiodomethane (4.6 mL, 56.5 mmol) in dichloromethane (14.6 mL) was added. After 10 minutes, a solution of diethyl (1S, 2S, 5R, 6S) -2-benzyloxycarbonylamino-4-methylene- bicyclo [3.1.0] hexane- (7.3 g, 18.8 mmol) in dichloromethane (5 mL) was added. After 1 hour, the ice bath was removed and the cloudy solution was allowed to stir at room temperature for 20 hours. After 24 h, the reaction mixture was quenched with cold 0.5 M aqueous hydrochloric acid (160 mL, 80 mmol) and dichloromethane (200 mL) and the mixture vigorously stirred. The dichloromethane layer was separated, then the dichloromethane layer was dried and filtered through two hydrophobic frites. Evaporated overnight to pale yellow oil and turned brown. Purification by flash chromatography eluting with ethyl acetate: iso-hexane (20:80) gave the unreacted diethyl (1S, 2S, 5R, 6S) -2-benzyloxycarbonylamino-4- methylene- bicyclo [3.1 0] hexane-2,6-dicarboxylate, the title compound (5.1 g) was obtained as a colorless oil.

A solution of trifluoroacetic acid (2.93 mL, 38.7 mmol) in dichloromethane (10 mL) was added to a solution of diethylzinc 1M (38.7 mL, 38.7 mmol) in heptane in dichloromethane (50 mL) Lt; / RTI > over 10 minutes. After 10 minutes, a solution of diiodomethane (3.12 mL, 38.7 mmol) in dichloromethane (10 mL) was added to the reaction mixture. After 10 minutes, a solution of the olefin and the diethyl (1S, 2S, 5R, 6S) -2-benzyloxycarbonylaminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane ] -2,6-dicarboxylate (5 g, 12.9 mmol) in tetrahydrofuran was added. After 1 hour the ice bath was removed and the cloudy solution was allowed to stir at room temperature for 16 hours. After 20 hours, the reaction mixture was quenched with cold 0.5 M hydrogen chloride (140 mL, 70 mmol) and dichloromethane (160 mL) and the mixture stirred vigorously. The dichloromethane layer was washed with 0.5 M aqueous hydrochloric acid (100 mL), dried over sodium sulfate, and then filtered through two hydrophobic frites. And evaporated to a light yellow oil (5.89 g). Purification by flash chromatography eluting with ethyl acetate: iso-hexane (15:85 to 25:75) afforded the title compound (4.28 g) as a colorless oil contaminated with small amounts of reactants.

Sodium bicarbonate (0.54 g, 6.45 mmol) and magnesium sulfate (0.78 g, 6.45 mmol) were redissolved in acetone (40 mL) and water (20 mL). Cool to 0 < 0 > C and add a bright orange potassium nitrate (0.2 g, 1.29 mmol) to give a light purple mixture. After 3 hours at room temperature, the solid was quenched with sodium thiosulfate pentahydrate (0.32 g, 1.29 mmol) and the suspension was filtered through a pad of diatomaceous earth and washed with acetone. Evaporation to a small volume, dilution with water (20 mL), and extraction with ethyl acetate (60 mL). The extract was washed with brine, dried, filtered and evaporated to an oil. Purification by flash chromatography eluting with ethyl acetate: iso-hexane (15:85 to 25:75) afforded the title compound (3.62 g, 70%) as a colorless oil.

Method 2:

A solution of 1M diethylzinc (1.6 L, 1.6 mol) in heptane was cannulated with dichloromethane (870.9 mL) at 0 < 0 > C (bath temperature -5 DEG C), then dichloromethane A solution of trifluoroacetic acid (121.02 mL, 1.6 mol) in tetrahydrofuran (870.9 mL) was slowly added. After 1 hour of addition, the mixture was stirred for 5 minutes, then diiodomethane (130.3 mL, 1.6 mol) was added in one portion and stirred for 15 minutes. To a solution of diethyl (1S, 2S, 5R, 6S) -2-benzyloxycarbonylamino-4-methylene-bicyclo [3.1.0] hexane-2,6-dicarboxylate (183.35 g) in dichloromethane (348.4 mL) g, 449.58 mmol) in THF (10 mL) over 10 min, and the mixture was stirred overnight at room temperature. Cooled to 0 < 0 > C, quenched with 0.5 M hydrochloric acid (1.5 L) and the mixture vigorously stirred. The organic layer was separated, washed with brine, and concentrated to dryness. Was dissolved in water (733.4 mL) and acetone (733.4 mL) and cooled to 0 C followed by magnesium sulfate (81.17 g, 674.37 mmol), sodium bicarbonate (56.65 g, 674.37 mmol) followed by 28.42 g , 179.83 mmol). The mixture was stirred at room temperature. After 30 minutes, sodium thiosulfate pentahydrate (197.10 g, 311.03 mmol) was added followed by diatomaceous earth (100 g). Stir for 30 minutes and filter through a pad of diatomaceous earth. The pad was washed with methyl-t-butyl ether and the aqueous layer was extracted with methyl-t-butyl ether. The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification by flash chromatography, eluting with hexane: methyl-t-butyl ether (10:90 to 50:50) afforded the title compound (92.67 g, 51%) as a colorless oil.

Spiro [bicyclo [3.1.0] hexane-4,1 ' -cyclopropane] -2, benzyloxycarbonyl (methyl) 6-dicarboxylate (22.67 g, 5.46 mmol).

Production Example 13

Diethyl (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate

Method 1:

(3.16 g, 37.2 mmol) was added to a solution of diethyl (1S, 2S, 5R, 6S) -2- (9H-fluoren-9- ylmethoxycarbonylamino) spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] 2,6-dicarboxylate (2.10 g, 4.29 mmol) at room temperature. After 30 minutes, it was evaporated to a yellow solid. Purification by flash chromatography eluting with ethyl acetate: iso-hexane (30:70 - > 80:20) eluted first 1- (9H-fluoren-9- ylmethyl) -piperidine and then the title compound The pale yellow liquid (1.07 g, 93%) was eluted.

Method 2:

To a solution of diethyl (1S, 2S, 5R, 6S) -2-benzyloxycarbonylaminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6- (3.62 g, 9 mmol) was dissolved and the solution was added to 10% palladium on carbon (Degussa type E101 NE / W, 0.18 g, 0.17 mmol). Hydrogenated on Parr equipment at 345 kPa for 4 hours. The reaction was filtered through a pad of diatomaceous earth to remove the catalyst, washed with ethanol and evaporated as the title compound (2.23 g, 93%) as a colorless oil.

Production Example 14

Amino] spiro [bicyclo [3.1.0] hexane-4, 2S, 2S, 5R, 6S) -2 - [[(2S) -2- (tert- butoxycarbonylamino) propanoyl] 1'-cyclopropane] -2,6-dicarboxylate

Cyclopropyl] -2,6-dicarboxylate (1.92 g, 7.17 mmol) was added to a solution of diethyl (1S, 2S, 5R, 6S) -2- aminospiro [bicyclo [3.1.0] hexane- (1.36 g, 10 mmol), 4-dimethylaminopyridine (9 mg, 717 μmol) and 1-hydroxybenzotriazole (1.36 g, 10 mmol) were combined in dichloromethane (48 mL). Was added triethylamine (1.5 mL, 10.8 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.92 g, 10 mmol) and stirred at room temperature for 2 hours under a nitrogen atmosphere . The reaction was diluted with ethyl acetate (200 mL), water (50 mL) and brine (50 mL). Stirred vigorously and then the ethyl acetate layer was separated and washed with 0.2 M hydrochloric acid (50 mL), water (50 mL), saturated sodium bicarbonate (50 mL) and brine (50 mL). The solution was dried, filtered and concentrated to give a yellow oil (3.48 g). Purification by flash chromatography eluting with ethyl acetate: iso-hexane (15:85 to 25:75) followed by (25:75 to 40:60) gave the material as an oil. Redissolved in dichloromethane, concentrated and dried under vacuum to give the title compound (3.05 g, 6.95 mmol) as a white foamy substance.

The following compounds were prepared essentially by the method of Preparation 14. < tb > < TABLE >

Production Example 17

Amino] spiro [bicyclo [3.1.0] hexane-4,1 ', 2', 5'- - cyclopropane] -2,6-dicarboxylic acid

Method 1:

A solution of 2M lithium hydroxide in water (27.8 mL, 55.6 mmol) in water was added dropwise to a solution of diethyl (1S, 2S, 5R, 6S) -2 - [[(2S) -2- (tert- Amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate (3.05 g, 6.9 mmol) in tetrahydrofuran Was added under nitrogen. The bilayer solution was stirred at room temperature for 20 hours. Acidified with 2 M hydrochloric acid (29 mL), ice (~ 20 g) and extracted with ethyl acetate (100 mL). The extract was washed with brine (50 mL), dried over sodium sulfate, filtered and evaporated to a white semi-solid foam. The material was redissolved in hot ethyl acetate (15 mL). Filtration and drying of the material in vacuo afforded the title compound (2.06 g, 5.4 mmol) as a white solid.

Method 2:

Diethyl (1S, 2S, 5R, 6S) -2-benzyloxycarbonylaminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate , 396.05 mmol) was dissolved in ethanol (792.1 mL) and palladium on carbon (15.9 g, 14.94 mmol) was added followed by a solution of 37.5% wt / wt hydrochloric acid in water (6.62 mL, 79.21 mmol). And hydrogenated at 413 kPa at room temperature on a 2 L Parr apparatus. After 4 hours the mixture was filtered through diatomaceous earth and a glass microfiber filter (Whatman) and concentrated in vacuo. The crude material was dissolved in tetrahydrofuran (396 mL) and chlorodimethoxytriazine (74.50 g, 415.86 mmol) and (2S) -2- (tert-butoxycarbonylamino) propanoic acid (79.88 g, 415.86 mmol ). The mixture was cooled to 0 C and N-methylmorpholine (131.06 mL, 1.19 mol) was added. The mixture was stirred at room temperature. After 5 hours the crude material was filtered through diatomaceous earth and the cake was washed with tetrahydrofuran (200 mL). The solvent was partially removed in vacuo and the resulting orange solution was cooled to 0 < 0 > C. 2 M sodium hydroxide in water (792.1 mL, 1.58 mol) was added dropwise. The mixture was allowed to stir overnight while allowing the reaction to reach room temperature. Dichloromethane (1 L) was added and the phases were separated. The organic layer was washed with additional water (300 mL). The combined aqueous layers were acidified with 1N potassium hydrogen sulfate to pH = 2-3 and then extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, filtered and evaporated in vacuo. The crude material was dissolved in tetrahydrofuran (600 mL) and heated to reflux. Heptane (2.1 L) was added and the solution was allowed to cool. The solid was filtered and dried in vacuo to give the title compound (149 g, 98%) as a white solid.

The following compounds were prepared essentially by Method 1 of Preparation 17. < RTI ID = 0.0 >

Production example 20

Diethyl (1S, 2S, 5R, 6S) -2- (tert-butoxycarbonylamino) spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate Late.

Method 1:

(1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate in dichloromethane (5 mL) (1.02 g, 3.61 mmol) followed by di-t-butyl dicarbonate (0.41 g, 1.88 mmol) in dichloromethane (6.00 mL) was added to a solution of diisopropylethylamine (0.47 g, 722.38 μmol) ) Was added and the reaction mixture was stirred overnight at room temperature. Additional di-t-butyl dicarbonate (0.32 g, 1.36 mmol) was added and stirred for an additional 2 h. Diluted with ethanol (20 mL) and purified by SCX-2 ion exchange resin cartridge (10 g) which had been pre-treated with two column volumes of ethanol. After loading, the cartridge was washed with four column volumes of ethanol and the eluent was concentrated in vacuo to give the crude product (808 mg). The crude material was purified by flash chromatography, eluting with ethyl acetate: cyclohexane (0: 100 → 40: 60) to give the desired material (100 mg). The cartridge was flushed with two column volumes of 3M ammonia in methanol to remove unreacted crude diethyl (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane ] -2,6-dicarboxylate as a yellow solid (430 mg). Di-t-butyl dicarbonate (0.32 g, 1.44 mmol) was added to a stirred solution of diethyl (1S, 2S, 5R, 6S) -2- aminospiro [bicyclo [3.1.0] Hexane-4,1'-cyclopropane] -2,6-dicarboxylate and stirred overnight at room temperature. Triethylamine (201.37 L, 1.44 mmol) and additional di-t-butyl dicarbonate (0.32 g, 1.44 mmol) were then added and stirring was maintained for 72 h. Di-t-butyl dicarbonate (0.32 g, 1.44 mmol) and catalyst N, N-dimethyl 4-aminopyridine (40.93 [mu] mol) were added and stirred overnight . The solution was diluted with ethanol (30 mL) and purified by SCX-2 ion exchange resin cartridge (10 g) which had been pre-treated with two column volumes of ethanol. Washed with four column volumes of ethanol, and the solution concentrated in vacuo to give 757 mg of the desired crude product. Purification by flash chromatography eluting with ethyl acetate: cyclohexane (0: 100 → 40: 60) gave the second fraction of the desired title product (24 mg). The two fractions were combined to give the title compound (337.47 [mu] mol, 47%).

Method 2:

Di-t-butyl dicarbonate (0.88 g, 4.01 mmol) was added to a solution of diethyl (1S, 2S, 5R, 6S) -2- amino- spiro [bicyclo [3.1.0] hexane- 4, 1 ' -cyclopropane] -2,6-dicarboxylate (1.4 g, 5.24 mmol) and the reaction mixture was stirred overnight at room temperature under nitrogen. Filter through a hydrophobic frit and wash the colorless gel with ethyl acetate. The combined organic layer was concentrated in vacuo to give a colorless oil. Purification by flash chromatography eluting with ethyl acetate: iso-hexane (0: 100 - > 30: 70) yielded 1.82 g of oil. Further drying under high vacuum gave the title compound (1.79 g, 93%).

Production Example 21

(1S, 2S, 5R, 6S) -2- (tert-butoxycarbonylamino) spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid

To a solution of diethyl (1S, 2S, 5R, 6S) -2- (tert-butoxycarbonylamino) spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate (1.79 g, 4.87 mmol) was added to a freshly prepared solution of lithium hydroxide monohydrate (1.64 g, 38.97 mmol) in water (19.5 mL) and stirred at 60 ° C overnight. Dilute with water (30 mL) and extract with ethyl acetate (2 x 50 mL). The aqueous and organic layers were separated. The organic layer was washed with 2M hydrochloric acid. The aqueous layer was washed with 2M hydrochloric acid (25 mL) and then with ethyl acetate (2 x 50 mL). The organic layers were combined and washed with brine (15 mL). Dried over magnesium sulfate, filtered, and concentrated to dryness. Redissolved in dichloromethane and concentrated to dryness to give the title compound (1.49 g, 98%).

Production Example 22

Dibenzyl (1S, 2S, 5R, 6S) -2- (tert-butoxycarbonylamino) spiro [bicyclo [3.1.0] hexane- 4,1'-cyclopropane] -2,6-dicarboxylate Rate

Cesium carbonate (0.24 g, 730.7 μmol) and benzyl bromide (87.16 μL, 730.7 μmol) were added to a solution of (1S, 2S, 5R, 6S) -2- (tert- Spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid (0.09 g, 292.29 [mu] mol). And stirred under nitrogen for 1.5 hours at room temperature. Quenched with water and extracted with ethyl acetate. The layers were separated and the organic portion was filtered through a hydrophobic frit and then concentrated to dryness to give the crude product (134 mg). Purification by flash chromatography eluting with ethyl acetate: iso-hexane (1:99 to 25:75) yielded a clear oil. Further purification by flash chromatography eluting with ethyl acetate: iso-hexane (1:99 to 10:90) afforded the title product (105 mg, 68%) as a clear oil.

The following compounds were prepared essentially by the method of Preparation 22.

Example 1

(1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid hydrochloride

5 mL of Reactivial was added to a solution of ditert-butyl (1S, 2S, 5R, 6S) -2- (tert- butoxycarbonylamino) spiro [bicyclo [3.1.0] hexane- Cyclopropane] -2,6-dicarboxylate (55 mg, 129.8 μmol). To this was added 5N aqueous hydrochloric acid solution (3 mL; 20.8 mmol) and 1,4-dioxane (1 mL). The mixture was stirred at 90 [deg.] C for 1 hour. Reactivial eggs were sealed and stirring was continued at 90 [deg.] C for 3 hours. Cooled to ambient temperature and allowed to stand for 3 days. And concentrated under reduced pressure to a dark solid. Oasis 占 HLB Waters (1 g) cartridge was prepared by washing with two column volumes of methanol followed by six column volumes of water. The dark solid was dissolved in water and loaded onto the cartridge. The cartridge was washed with water (2 column volumes) and the eluent was collected. The solution was freeze-dried to give the title compound (16.1 mg, 65 [mu] mol).

Example 2

(1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid

Diethyl (1S, 2S, 5R, 6S) -2-acetamidospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate mmol) was added 2M sodium hydroxide (11.5 mL, 23.1 mmol). Upon addition, the reaction mixture was heated to reflux under a blanket of nitrogen. After 21 hours, excess 2M sodium hydroxide (5.8 mL, 11.5 mmol) was added and heating was resumed for 120 hours. And purified by cation exchange chromatography (Dowex (TM) 50X8-100) as follows. Any insoluble particles are filtered off and rinsed with HPLC grade water. The solution was concentrated in half. The solution was loaded onto the resin while flowing through the column at a drop rate of about 1 drop per 1-2 seconds. After the initial loading volume was spotted on the resin surface, the resin was rinsed with HPLC grade (~ 1 to 2 column volumes) and this was repeated three times. The pH of the eluate is monitored and rinsed with HPLC grade until the application is complete (pH return to pH = 7 again). Once a complete pH cycle was observed and the eluate returned to pH = 7, the column was washed with more than one column volume each of HPLC grade, HPLC grade: tetrahydrofuran (1: 1) followed by HPLC grade. The product was replaced with 10% Pyridine: HPLC grade water from the resin and elution continued with 10% Pyridine: HPLC grade water until no further product was detected by TLC. Fractions containing the desired material were combined and concentrated to dryness to yield a white solid. Freeze-dried to give the title compound (795 mg, 3.8 mmol).

Example 3

(1S, 2S, 5R, 6S) -2 - [[(2S) -2-aminopropanoyl] amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] - dicarboxylic acid; 1,4-dioxane (1: 0.5); Hydrochloride.

A solution of 4M hydrogen chloride (1.57 mL, 6.3 mmol) in 1,4-dioxane was added to a solution of (lS, 2S, 5R, 6S) -2- [ propyl] amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid (0.16 g, 418.4 μmol) Was added to the stirred suspension under nitrogen at room temperature. After 16 hours the white solid was filtered off, washed with 1,4-dioxane and dried at 60 < 0 > C under vacuum overnight to give the title compound (0.14 g).

Example 4

(1S, 2S, 5R, 6S) -2 - [[(2S) -2-aminopropanoyl] amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] - dicarboxylic acid

(36.94 mL, 430.11 mmol) was added to a solution of (1S, 2S, 5R, 6S) -2 - [[(2S) -2- (tert- butoxycarbonylamino) propanoyl] amino ] Spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid (82.24 g, 215.06 mmol) and the mixture was stirred at 50 ° C. After 1.5 hours the mixture was cooled to 0 C and 50% sodium hydroxide in water was added at pH = 3.6-3.8. The resulting solid was stirred for 1 hour, filtered and washed with water. Drying under vacuum for 48 hours afforded the title compound (36 g, 127.53 mmol) as a white solid.

Example 5

(1S, 2S, 5R, 6S) -2 - [[(2S) -2-aminopropanoyl] amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] - dicarboxylic acid hydrochloride

4M HCl in 1,4-dioxane (20 mL, 80 mmol) was added to a solution of (1S, 2S, 5R, 6S) -2 - [[(2S) -2- Propyl] amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid (2.06 g, 5.4 mmol) Was added to the suspension under nitrogen at room temperature. The reaction mixture was sonicated for several minutes and then stirred for 16 hours. The solid was filtered, washed with 1,4-dioxane and dried under vacuum at 6O < 0 > C for 6 hours to give 2.2 g of a white solid. The material was redissolved in HPLC grade (20 mL), filtered to remove any insoluble particles, and the filtrate was freeze-dried to give the title compound (1.51 g, 4.75 mmol).

The following compounds were prepared essentially by the method of Example 4. [

Example 8

Dibenzyl (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate; 1,4-dioxane; Hydrochloride

A solution of dibenzyl (1S, 2S, 5R, 6S) -2- (tert-butoxycarbonylamino) ) Spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate (0.1 g, 0.2 mmol) at room temperature under nitrogen, Lt; / RTI > An excess of 4M hydrogen chloride solution (493.30 L, 1.97 mmol) in 1,4-dioxane was added and heated to 80 < 0 > C. Concentrated to dryness, triturated with acetonitrile and the suspension was freeze-dried to give the title compound (89.9 mg, 88%) as a white solid as a (1: 1) adduct with 1,4-dioxane.

Example 9

Bis [(2-fluorophenyl) methyl] (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] Foxylate hydrochloride

백색 고체를 아세토니트릴 (8 mL) 중에 용해시켜 반투명 용액을 수득하였다. 1시간 동안 정치되도록 한 후, 여과하였다. 여과물을 진공 하에 농축시켜 점착성 발포체를 수득하였다. 에틸 아세테이트 중에 재용해시키고, 탄산수소나트륨의 포화 용액으로 세척하고, 황산마그네슘 상에서 건조시키고, 여과하고, 진공 하에 농축시켜 774 mg을 수득하였다. 디에틸 에테르 (11 mL) 중에 재용해시키고, 디에틸 에테르 중의 1M 염화수소 (1.86 mL, 1.86 mmol)를 첨가하고, 진공 하에 농축시켜 백색 고체 발포체를 수득하였다. 진공 하에 50℃에서 추가로 밤새 건조시켜 표제 화합물 (0.74 g, 85%)을 수득하였다.

A solution of bis [(2-fluorophenyl) methyl] (1S, 2S, 5R, 6S) - (4-chlorophenyl) To a stirred solution of 2- (tert-butoxycarbonylamino) spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate (0.98 g, 1.86 mmol) At room temperature under nitrogen, and the solution was stirred for 20 hours. Concentration in vacuo yielded oil (0.93 g). Was dissolved in a mixture of acetonitrile (10 mL) and water (30 mL) and lyophilized over the weekend to give (lS, 2S, 5R, 6S) -2- amino- 2- [(2- fluorophenyl) methoxy Carbonyl] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -6-carboxylic acid (~7-10%) as a white solid (820 mg) .

The white solid was dissolved in acetonitrile (8 mL) to give a translucent solution. Allowed to stand for 1 hour, and then filtered. The filtrate was concentrated in vacuo to give a viscous foam. Redissolved in ethyl acetate, washed with a saturated solution of sodium hydrogencarbonate, dried over magnesium sulfate, filtered and concentrated in vacuo to give 774 mg. Redissolved in diethyl ether (11 mL), 1 M hydrogen chloride in diethyl ether (1.86 mL, 1.86 mmol) was added and concentrated in vacuo to give a white solid foam. Further drying overnight under vacuum at 50 <0> C gave the title compound (0.74 g, 85%).

Example 10

(1S, 2S, 5R, 6S) -2 - [[(2S) -2-aminopropanoyl] amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] - dicarboxylic acid dihydrate

Step 1: Diethyl (1S, 2S, 5R, 6R) -2-amino-4-oxo-bicyclo [3.1.0] hexane-2,6-dicarboxylate hydrochloride

Under nitrogen atmosphere, acetyl chloride (86.5 mL, 1.2 mol) was added dropwise to absolute ethanol (1.0 L, 17.2 mol) while maintaining the internal reaction temperature below 30 &lt; 0 &gt; C. The resulting mixture was stirred for 15 min and a solution of di-t-butyl (lS, 2S, 5R, 6R) -2- (tert- butoxycarbonylamino) -4-oxo-bicyclo [3.1.0] hexane- 2,6-dicarboxylate (100 g, 0.24 mol) was added in one portion. The resulting mixture was heated to reflux for 16-20 hours. The reaction mixture was concentrated to an oil under reduced pressure. The crude product was dissolved in methylene chloride (250 mL) and concentrated in vacuo. The methylene chloride / concentration process was repeated to give a white foam. Ethyl acetate (150 mL) was added and the mixture was heated to 65 &lt; 0 &gt; C. Methyl t-butyl ether (100 mL) was added and the mixture was stirred at 65 [deg.] C for 15 minutes. Methyl t-butyl ether (300 mL) was added over 15 minutes and stirred at 65 &lt; 0 &gt; C for 15 minutes, then heating was discontinued and the slurry allowed to cool to ambient temperature. The mixture was filtered and the filter cake was washed with methyl t-butyl ether (150 mL). The cake was transferred to a vacuum oven and dried overnight at 25 &lt; 0 &gt; C to give the crude product (67.5 g). The solid was transferred to a round bottom flask, diluted with ethyl acetate (170 mL) and the mixture was heated to 65 &lt; 0 &gt; C. The mixture was stirred for 1 hour, and tetrahydrofuran (68 mL) and ethanol (3 mL) were added. The heat source was stopped and methyl t-butyl ether (272 mL) was added over 20 minutes while allowing the mixture to cool to ambient temperature. The slurry was filtered and the cake washed with 95/5 methyl t-butyl ether / ethyl acetate (2 x 75 mL) and further dried in a vacuum oven at 25 &lt; 0 &gt; C overnight to give the title compound , 98.0%).

Step 2: Diethyl (1S, 2S, 5R, 6R) -2-benzyloxycarbonylamino-4-oxo-bicyclo [3.1.0] hexane-2,6-dicarboxylate

Bicyclo [3.1.0] hexane-2,6-dicarboxylate-hydrochloride (55.0 g, 188.5 mmol) was added to a solution of tetra Was suspended in hydrofuran (220 mL), followed by water (220 mL) and potassium carbonate (92.1 g, 659.9 mmol). The resulting mixture was stirred for 30 minutes. Benzyl chloroformate (26.7 mL, 175.3 mmol) was added to the mixture over 45 minutes while maintaining the reaction temperature below 25 &lt; 0 &gt; C. The reaction mixture was stirred for 30 minutes, then diluted with ethyl acetate (550 mL) and water (275 mL). The phases were separated and the aqueous layer was back extracted with ethyl acetate (250 mL). The organic portion was combined and washed sequentially with aqueous HCl (0.5 N, 100 mL), saturated NaHCO ₃ (100 mL) and brine (100 mL). The organic solution was dried over Na ₂ SO ₄ , filtered and concentrated in vacuo to give the title compound (67.6 g, 92.1%) as a clear oil.

Step 3: Diethyl (lS, 2S, 5R, 6S) -2-benzyloxycarbonylamino-4-methylene- bicyclo [3.1.0] hexane- 2,6-dicarboxylate

Under a nitrogen atmosphere, methyl-triphenylphosphonium bromide (67.4 g, 184.9 mmol) and tetrahydrofuran (300 mL) were added with stirring. A solution of potassium tert-butoxide (1M) (184.9 mL, 184.9 mmol) in tetrahydrofuran was added to the reaction mixture over 15 minutes and the resulting slurry was stirred for 3 hours at ambient temperature. (12.3 kg, 31.6 mol) of diethyl (1S, 2S, 5R, 6R) -2-benzyloxycarbonylamino- Was dissolved in hydrofuran (120 mL) and the reaction mixture was added over 1 hour while maintaining the reaction temperature below 30 &lt; 0 &gt; C. The slurry formed at ambient temperature was stirred overnight, then diluted with ethyl acetate (600 mL) and quenched with water (300 mL). After stirring the biphasic mixture for 30 minutes, the layers were separated and the organic portion washed with water (300 mL) followed by 0.25 M HCl (300 mL). The organic portion was dried over Na ₂ SO _4, filtered, and concentrated in vacuo to give the (45 ℃), as a dark oil crude product (111.0 g). Purification of the material by silica gel plug chromatography (1 Kg Kieselgel-60, 4 L 15% EtOAc in heptane, then 10 L of 30% EtOAc in heptane) gave the title compound (37.3 g, 87.9% Concentration, 54.9%).

Step 4: Preparation of diethyl (1S, 2S, 5R, 6S) -2-benzyloxycarbonylaminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate

To the jacketed reaction flask connected to the cooler unit was added diethylzinc solution (157.2 mL, 157.2 mmol, 1M in heptane) under a nitrogen atmosphere. The solution was cooled to -15 C and diluted with cold (-15 C) dry dichloroethane (157.2 mL). Trifluoroacetic acid (11.1 mL, 146.7 mmol) was dissolved in dichloroethane (11.1 mL) and added to the reaction vessel over 50 minutes maintaining the internal temperature below -10 &lt; 0 &gt; C. The resulting suspension was stirred at -10 to -15 &lt; 0 &gt; C for 30 minutes. Diiodomethane (12.7 mL, 42.1 g, 157.2 mmol) was dissolved in dichloroethane (12.7 mL) and added to the reaction mixture over 50 minutes while keeping the internal temperature below -10 &lt; 0 &gt; C. The resulting dilute white suspension was stirred at -10 to -15 &lt; 0 &gt; C for 30 minutes. A solution of diethyl (1S, 2S, 5R, 6S) -2-benzyloxycarbonylamino-4-methylene-bicyclo [3.1.0] hexane-2,6-dicarboxylate (20.3 g, 87.9% mmol) was dissolved in dichloroethane (30.4 mL) and added to the reaction mixture over 10 minutes while keeping the internal temperature below -10 &lt; 0 &gt; C. The clear, light yellow solution was stirred at -10 &lt; 0 &gt; C for 5 minutes and the latent heat was monitored. The set point on the chiller was changed to 0 ° C and the reaction mixture was stirred at this temperature for 48 hours. The reaction mixture was quenched by the addition of 5N HCl (62.9 mL, 314.4 mmol) over 15 minutes, keeping the internal temperature below 6 &lt; 0 &gt; C. The resulting mixture was stirred at 0-5 &lt; 0 &gt; C for 20 minutes, then transferred to a funnel and the layers were separated. The organic layer was washed with 1 N HCl (2 x 50 mL) followed by 1: 1 saturated NaHCO ₃ / H ₂ O (60 mL), 1: 1 saturated Na ₂ CO ₃ / H ₂ O (60 mL) Lt; / RTI &gt; The organic portion was dried over Na ₂ SO _4, was obtained as a pale yellow oil and concentrated under vacuo to give the title compound (19.6 g, 60.3% concentration, 63.9% corrected yield).

Step 5: Synthesis of diethyl (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate hydrochloride

(100 mL, 10 vol), concentrated HCl (2.1 mL, 1.0 N) and diethyl (1S, 2S, 5R, 6S) -2- benzyloxycarbonylaminospiro [bicyclo [3.1.0] hexane- 4,1'-cyclopropane] -2,6-dicarboxylate (9.7 g, 24.16 mmol) followed by Pd black (3.9 g, 40% by weight) was charged to the HEL reactor. The reactor was purged 3 times with nitrogen followed by 3 times with hydrogen and pressurized with 40 psi of hydrogen. The reaction was allowed to stir at 20-30 &lt; 0 &gt; C for 2 hours or more until completion of the reaction was monitored by HPLC. The resulting slurry was filtered through a pad of Hyflo Super Cel (R) and the wet cake was washed with absolute ethanol (2 x 30 mL, 3 vol.). The filtrate was transferred to a clean reactor and approximately 5 volumes of ethanol were replaced with isopropyl acetate based on the in-system product yield from the calibration curve. The resulting slurry was cooled to &lt; RTI ID = 0.0 &gt; 0-5 C &lt; / RTI &gt; The resulting slurry was filtered and the wet cake washed with cold isopropyl acetate (3 x 5 mL, 0.5 volume) was washed. Drying at 30 [deg.] C under reduced pressure for at least 12 h gave the title compound (4.66 g, 15.34 mmol, 63.5%).

Step 6: Diethyl (1S, 2S, 5R, 6S) -2 - [[(2S) -2- (tert- butoxycarbonylamino) propanoyl] amino] spiro [bicyclo [3.1.0] hexane -4,1'-cyclopropane] -2,6-dicarboxylate

N-methylmorpholine (1.96 mL, 1.2 eq.) Was added followed by tetrahydrofuran (45 mL, 10 vol) and (2S) -2- (tert- butoxycarbonylamino) propanoic acid 2-Chloro-4,6-dimethoxy-1,3,5-triazine (3.12 g, 1.2 eq.) Was charged to the reactor. The resulting dilute slurry was stirred at 20-25 [deg.] C for 3 hours or more until the reaction was completed by HPLC. Cyclopentane] -2,6-dicarboxylate hydrochloride (4.55 g, 14.81 &lt; RTI ID = 0.0 &gt; mmol) were charged at a time at 20-25 ° C, and then N-methylmorpholine (1.63 mL, 1.0 eq.) was charged over 10 minutes while maintaining the temperature at 20-25 ° C. The resulting slurry was stirred at 20-25 [deg.] C for 2 hours or more until the reaction was completed by HPLC. The resulting solid was filtered and the wet cake was washed with tetrahydrofuran (2 x 10 mL, 2.2 vol) to yield the title compound assuming 100% yield. A pale yellow amorphous tetrahydrofuran solution of the title compound was used without further purification assuming 100% yield.

Step 7: (1S, 2S, 5R, 6S) -2 - [[(2S) -2- (tert- Butoxycarbonylamino) propanoyl] amino] spiro [bicyclo [3.1.0] , 1'-cyclopropane] -2,6-dicarboxylic acid

Amino] spiro [2S, 2S, 5R, 6S) -2 - [[(2S) -2- (tert- butoxycarbonylamino) propanoyl] (6.50 g, 14.81 mmol) was charged to the reactor and the reaction was allowed to proceed for 12 hours RTI ID = 0.0 &gt; 20-30 C. &lt; / RTI &gt; The reaction was transferred to a separatory funnel and allowed to settle for at least 10 minutes. The phases were separated and the lower aqueous layer was returned to the reactor. Ethyl acetate (90 mL, 13.8 vol) to the mixture, followed by a 1 N NaHSO ₄ was added until the pH reached 2 to 2.5. The layers were separated and the organic portion washed with water (45 mL, 6.9 vol). The water in the organic phase was removed by atmospheric distillation with ethyl acetate to remove residual water. The resulting slurry was cooled at 20-30 ° C over 2 hours and allowed to granulate at this temperature for at least 90 minutes. The resulting solid was filtered and the wet cake was washed with ethyl acetate (3 x 15 mL, 2.3 vol.). Drying at 45 [deg.] C under reduced pressure gave the title compound (4.45 g, 11.64 mmol, 78.6%) as a white solid.

Step 8: (1S, 2S, 5R, 6S) -2 - [[(2S) -2-Aminopropanoyl] amino] spiro [bicyclo [3.1.0] hexane- 2,6-dicarboxylic acid dihydrate

Amino] spiro [bicyclo [3.1.2 (3H) -quinolinone &lt; / RTI &gt; (3.99 g, 10.4 mmol) followed by conc. HCl (1.80 mL, 2.0 eq.) Was charged to the reactor and then the reaction was complete (Monitored by HPLC) and the reaction was heated to 45-55 [deg.] C. The reaction mixture was cooled to 20-30 &lt; 0 &gt; C and the pH was adjusted to approximately 3.6 using 5 N NaOH. Anhydrous ethanol (15 mL, 3.75 vol) was added over 30 minutes to the resulting slurry at 20-30 &lt; 0 &gt; C. The resulting slurry was allowed to granulate at &lt; RTI ID = 0.0 &gt; 20-30 C &lt; / RTI &gt; The mixture was cooled to -5-5 &lt; 0 &gt; C and allowed to granulate for more than 60 minutes. The resulting solid was filtered and the cake washed with 30% absolute ethanol in water (2 x 9 mL, 2.25 vol). The solid was dried under reduced pressure at 35 &lt; 0 &gt; C for 12 hours and then the resulting solid was kept on the balance until there was no further weight change for more than 2 hours to yield the title compound (2.71 g, 8.51 mmol, 81.6% &Lt; / RTI &gt;

The X-ray powder diffraction (XRD) pattern of the crystalline solid was measured on a Bruker D4 Endeavor X-ray with a CuKa source (lambda = 1.54060 A) operating at 35 kV and 50 mA and a Vantec detector Powder diffractometer. This sample was subjected to a scanning electron microscope at 4 to 40 DEG C using a 0.6 mm diverging slit, a 5.28 mm fixed scattering prevention slit and a 9.5 mm detection slit using a step size of 0.0087 DEG (2?) And a scanning speed of 0.5 sec / 2 &amp;thetas;). The dry powder was packed onto a quartz sample holder and a smooth surface was obtained using a glass slide. The diffraction pattern was adjusted based on the NIST 675 standard peak at 8.85 and 26.77 degrees 2-theta. In the case of any given crystal form, it is well known in crystal chemistry that the relative intensities of the diffraction peaks can be varied due to the preferred orientation due to factors such as crystal habits, and the angular peak positions can vary slightly. For example, the peak position may shift due to changes in temperature or humidity analyzing the sample and sample displacement. In this case, the peak position variability +/- 0.2 (2 [theta]) will consider these potential changes without interfering with the obvious confirmation of the indicated crystal form. Confirmation of the crystal form can be made on the basis of characteristic peaks (in units of 2 &amp;thetas;), typically any unique combination of more pronounced peaks. The crystal form diffraction pattern was collected at ambient temperature and relative humidity.

Thus, the sample produced in Example 10 is characterized by an XRD pattern using CuKa radiation with a diffraction peak (2-theta value) as described in Table 1 below. Specifically, the pattern contains peaks at 5.20 in combination with at least one of the peaks selected from the group consisting of 10.45, 11.70, 15.75, 21.06 and 23.59 with a tolerance for the diffraction angle of 0.2 degrees.

Table 1 X-ray powder diffraction peak of Example 10

The mGlu receptor is a G-protein-coupled receptor that modulates neuronal excitability. More particularly, the altered glutamate neurotransmission is associated with neurological disorders such as chronic pain conditions such as persistent pain, neuropathic pain, chronic inflammatory pain or visceral pain; Psychotic disorders such as schizophrenia, bipolar disorder, generalized anxiety disorder or post-traumatic stress disorder; Or neurodegenerative disorders.

Since the compounds of the present invention are mGlu2 / 3 agonists, they may be suitable for treating the above-mentioned conditions.

Human mGlu2 and mGlu3 agonist FLIPR assay

Derived AV-12 cell line derived from Syrian Hamster fibroblasts and stably expressing the human mGlu2 or mGlu3 receptor and co-transfected with rat glutamate transporter EAAT 1 (excitatory amino acid transporter 1) and G? 15 subunits Were used for the study. Expression of G? 15 causes the G-coupled receptor to signal through the phospholipase C pathway, thus producing the ability to measure receptor activation by fluorescent assay calcium assay assays. Cell lines were treated with 5% dialyzed fetal bovine serum, 1 mM sodium pyruvate, 10 mM HEPES (4- (2-hydroxyethyl) -1-piperazinethanesulfonic acid), 1 mM L-glutamine and 5 [ (DMEM) containing high glucose and pyridoxine hydrochloride supplemented with dextrose (all media were purchased from Invitrogen). The full grown cultures were transfected biweekly using an enzyme-free dissociation solution (Chemicon S-004-B). Cells were harvested 24 h prior to assay and cultured in media containing only 250 (mGlu2) or 125 (mGlu3) μM L-glutamine (freshly added) using a Matrix Well-Mate cell- (MGlu2) or 115,000 (mGlu3) cells per well in a -well black-wall poly-D-lysine-coated plate (BD BioCoat # 354640). Intracellular calcium levels were monitored before and after compound addition using a fluorescence imaging plate reader (FLIPR, Molecular Devices). The assay buffer consisted of Hank's buffered saline solution (HBSS; Sigma) supplemented with 20 mM HEPES. The medium was removed and cells were incubated with 8 μM Fluo-3 AM (Molecular Probes, F-1241; 50 μL per well) in assay buffer for 90 min (mGlu2) or 120 min ). &Lt; / RTI &gt; The dye solution was removed and replaced with a new assay buffer (50 μL per well). Single-addition FLIPR assays were performed prior to each experiment to generate a 11-point concentration response curve (starting at 10 uM with 3X dilution) to the agonist glutamate (Fisher A 125-100) to confirm a typical EC ₅₀ response Respectively. The results were analyzed using Prism v4.03 (GraphPad Software). Compounds of the invention were tested in single-dose FLIPR assays starting at a final concentration of 25 [mu] M using a 10-point concentration response profile using 3X dilution. The compounds of the invention were solubilized as 10 mM stock in 0.1 N NaOH and stored at -20 &lt; 0 &gt; C. They were diluted in black buffer through a triple dilution series. After the initial 5-second fluorescence read out on the FLIPR instrument was obtained, the compound of the present invention was added to the cell plate (50 μL per well). To detect agonist activity, data was collected every second for the first 30 seconds and then every 3 seconds for a total of 90 seconds. The maximal response was defined as being induced by ECmax (100 [mu] M glutamate). The compound effect was measured as the maximum minus minimum peak height in relative fluorescence units (RFU) corrected for the reference fluorescence measured in the absence of glutamate. Crystals were performed using a single plate. Agonist efficacy was quantified as the percent stimulation induced by the compound alone in relation to the maximal glutamate response. All data were calculated as relative EC ₅₀ values using a four-variable logistic curve fitting program (ActivityBase v5.3.1.22).

Example 2 obtained EC ₅₀ (n = 3, error calculated as SEM) of 39.0 nM +/- 5.9 as measured by the hmGlu2 FLIPR assay as practiced above. Example 2 also yielded an EC ₅₀ (n = 8, error calculated as SEM) of 285 nM ± 52.5 as measured by the hmGlu3 FLIPR assay as practiced above. These results demonstrate that Example 2 is a potent mGlu2 and mGlu3 agonist.

Reversal of persistent pain induced by formalin injection

Administration of formalin to the plantar surface of the hind foot of a rat results in two steps of invasive protective action (e.g., licking, bite and jerking of injected foot): an initial stage of about 5 minutes after formalin administration, and about 10 Minute to 60 minutes later. A quiescent period of approximately 5 minutes to 10 minutes was separated into two steps. Scoring of these formalin-induced behaviors could be automated using stimulus chambers (SR-Lab, San Diego Instruments, San Diego, Calif.) To detect movement of the rats by an accelerometer. Formalin was injected intraplantarly 1 hour after administration of the test compound (active) within 0.3-10 mg / kg (intraperitoneal route) in non-fasted male Sprague-Dawley rats. The rats were then placed individually in the cylinders in the test chamber for compliance. Formalin was injected intravenously 2 hours after oral administration of the test compound (prodrug) in the range of 0.45-15 mg / kg in non-fast male Sprague-Dole rats. The rats were then placed individually in the cylinders in the test chamber for compliance. At some point, the rats were removed from the cylinder and subsequently formalin (50 μL of 5% solution in saline) was administered into the plantar surface of the right hind paw and immediately placed in the cylinder again. The cylinder was placed on the loading cell of the detection system in the test chamber, so that the reaction was continuously monitored for 60 minutes at 1 second. The number of infringing defensive events [> 20 - the number of 1-second bins with loading units] were added in 5-minute intervals. The 20-loading unit threshold is large enough to remove normal physiological events, such as breathing or sniffing, but is an important scale for detecting infringing defensive events. Over 60 minutes of data collection, the data was transformed by determining the number of events over a threshold (20 loading units) in each one second time bin. The initial score was the sum of events of more than 20 loading units at time 0 to 5 minutes. The late-stage score was obtained by adding the total number of events of more than 20 loading units in the 11th to 40th data collection period. Formalin data was assessed by one-way analysis of variance (ANOVA) and appropriate controls were compared using a JMP (v 6.0.2) statistical analysis program (SAS Institute Inc., Carrie, NC) Dunnett's 't' test. The difference was considered significant when the p-value was less than 0.05. ED ₅₀ calculations were performed using non-linear regression curve fitting on the reversed-transformed data percent for each dose.

Example 2 gave an ED ₅₀ of 0.7 mg / kg (ip) as measured in this assay, which was practiced as described above. Example 10 gave an ED ₅₀ of 4.8 mg / kg (po) as measured in this assay, which was practiced as described above. These results demonstrate that the compounds within the scope of the present invention are useful drugs for persistent pain.

Inversion of L5 / L6 nerve ligation induced tactile allodynia

Unilateral ligation of nerve-stimulated nerves in the hind legs resulted in chronic persistent pain, which appeared in tactile allodynia in rats. L5 / L6 neuronal ligation was performed (Kim et al. Pain (1992) 50, 355-363). Nerve damage was generated by firmly ligation of left L5 and L6 spinal nerves under gas phase anesthesia with a mixture of isoflurane (3% for induction and 2% for retention) and oxygen. At least 14 days after surgery, tactile allodynia was assessed by measuring the tactile sensitivity of the injured foot to the von Frey filament with incremental bending force (0.3-15 g) (Chaplan et al., J. of Neuroscience Methods (1994) 53 , 55-63). When the rat proved tactile allodynia (foot palsy in response to application of a bending force of less than 2 grams), they were considered hypersensitive. The baseline value was determined just before the test compound was evaluated. The test compound (prodrug) was orally administered at 15 mg / kg and the tactile threshold for foot evasion was measured at 1, 2 and 4 hours after administration. Data were expressed as reaction thresholds (grams (g)), and the values had an average standard error (± SE average) for each time point. Data were analyzed using Dunnett post-mortem analysis followed by analysis of variance followed by an absolute change in pain threshold (Cmax = maximal response baseline) and an average [log (maximal response) -log (pre-dose score)] Respectively.

Example 10, which was orally administered at 15 mg / kg, wherein n = 8 (error calculated as SEM), was measured in this assay as practiced as described above to determine the mechanical threshold after vehicle treatment (9.06 ± 2.26, 12.31 ± 1.86 and 8.63 ± 1.98 at 1, 2 and 4 hours, respectively) as compared to the control group (1.50 ± 0.60, 1.34 ± 0.75 and 2.35 ± 0.62 at time). These results demonstrate that compounds within the scope of the present invention are useful drugs for neuropathic pain conditions.

Inversion of CFA-induced mechanical hyperalgesia

The induction of local inflammation in the hind feet of the rats resulted in persistent mechanical hyperalgesia that could be measured by determining the threshold for pressure stimulation to give a painful response. The method for complete Freund's adjuvant (CFA) inducing mechanical hyperalgesia in rats is largely described in Iadarola et al. Brain Res. (1988) 455, 205-212. The rats were placed under isoflurane anesthesia and injected into the right foot with 50 μl of CFA (Sigma C5881, 85% paraffin oil, 1 mg / ml Mycobacterium extract in 15% mannide monooleate) in the foot. Animals were restored in a soft litter cage and mechanical hyperalgesia changes were measured 24 hours after CFA injection. Mechanical hyperalgesia (Randall Sellito Test) was determined by carefully stopping the rats and placing the feet between flint and pusher (Ugo Basile Analgesy-meter). The gram force was recorded when the animal avoids the foot. A maximum force of 250 grams or less was applied and a value of 250 grams was recorded if the rat did not avoid footing.

Rats were tested for baseline response before CFA injection. After CFA, the reaction was tested the following morning (approximately 24 hours after injection of CFA). Rats were randomized on a response to CFA after randomization with random rats showing scores of > 150 grams removed from further testing. The test compound (prodrug) was orally administered in the range of 1.5 to 15 mg / kg and the mechanical threshold for foot palsy was measured at 1 and 2 hours after administration. Statistical significance was defined as a p-value <0.05 in post Dunnett's test following covariance analysis for each time point. Table 2 below provides statistically significant results for Example 10 which was practiced as described above. These results demonstrate that the compounds within the scope of the present invention are useful medicaments for chronic inflammatory pain conditions such as osteoarthritis or rheumatoid arthritis.

<Table 2>

Values are mean ± SEM for N = 8-10 per group.

* P value at the same time point <0.05 vs. Vehicle (ANOVA and Dunnett t-test)

Colon rectal bloating - reversal of induced pain behavior.

The rats' internal cask was caused by the bulging of the rectum rectum, and the pain was monitored by approaching the reflex contraction of the abs. Measurement of abdominal reflex contractions with pain caused by colonic rectal bloating in rats is described in Fioramonti et al. Neurogastroenterology and Motility (2003) 15, 363-369, and Urban et al. J. of Pharmacology and Exp. Ther. (1999) 290, 207-213. Male Sprague Dawley rats were implanted surgically using electromyographic (EMG) electrodes in the abdominal cavity and restored for one week, during which the animals were adhered to handling and partial control to minimize the effect of stress. After inserting a lubricated latex balloon attached to a pressure monitor / regulator staged at 20, 40, and 60 mm Hg pressure for 20 seconds duration with a 3 minute dwell period between runs, the baseline EMG measurement Were collected and recorded. After recording a set of three exposures at each pressure, the pressure was stepped to the next level. Data were collected under curves (μV per second) for EMG readout during pressure stimulation and averaged over three trials. Colonic rectal distention was initiated 90 minutes after oral administration of the test compound (prodrug) within the range of 1.5 to 17 mg / kg. Statistical significance was defined as a p-value <0.05 in Dunnett's post-test following covariance analysis. Table 3 below provides statistically significant results for Example 10 which was practiced as described above. These results demonstrate that compounds within the scope of the present invention are useful drugs for visceral conditions.

<Table 3>

Values are mean ± SEM for N = 8-16 per group.

* P value at the same time point <0.05 vs. Vehicle (ANOVA and Dunnett t-test)

Inhibition of phencyclidine (PCP) -induced and motor activity in rats

Administration of an NMDA receptor antagonist, such as ketamine or phencyclidine (PCP), produced a psychotic-like effect in humans similar to those observed in patients with schizophrenia. The ability of agonists to reverse the exercise-stimulating effects of NMDA antagonists is often used as an animal model of psychosis, which has demonstrated excellent predictability for detecting clinical efficacy of medicines for schizophrenia and bipolar disorder.

(Harlan, Indianapolis, IN) in a clear plastic shoe box cage of dimensions 45 x 25 x 20 cm with a 1 cm deep wood chip as the litter and a metal grill on top of the cage. Exercise activity was monitored by placing the rats. The motion monitor (Kinder Scientific) is a rectangular rack of 12 photo beams arranged in 8 x 4 format (or 22 high density groupings in a 15 x 7 pattern) 5 cm high, and a second rack (To measure breeding behavior). The shoe box cage was placed inside these racks, where the racks were placed in a 3-foot-high table top in a separate room. The test compound was administered at a dose of 5 mg / kg of phencyclidine (PCP) 30 minutes after administration of the compound of the present invention (active) within the range of 0.3-10 mg / kg (intraperitoneal route). The compound (prodrug) of the present invention was orally administered to overnight fasted rats within the range of 0.3 - 30 mg / kg. After 4 hours, 5 mg / kg of PCP was administered. On the day of the test, the rats are placed in a test cage and allowed to acclimate for 30 minutes before administration of the PCP test; The rats were monitored for an additional 60 minutes after PCP administration.

Data analysis and ED ₅₀ calculations were performed using GraphPad Prism (San Diego, Calif., USA). The power analysis determined that 8-10 rats per group were required to have appropriate statistical power to detect treatment differences (power = 0.8). One-way analysis of variance (ANOVA) with a post-Dunnett multiple comparison test was performed on total 60-minute motor activity. ED ₅₀ calculations were performed using non-linear regression curve fitting on the reversed-transformed data percent for each dose.

Example 2 obtained ED ₅₀ of 1.46 mg / kg (ip) as measured in this assay as practiced above. Examples 5 and 6 obtained 2.95 mg / kg (po) and 4.31 mg / kg (po), respectively, as measured in the actual assay as described above. These results demonstrate that compounds within the scope of the present invention are useful drugs for schizophrenia and bipolar disorder.

Stress-induced weakening of high fever in rats

High fever (a rise in deep body temperature) is a common phenomenon that has been well proven in response to stress in many mammals, including humans. In many anxiety disorders, hyperthermia occurs as part of the pathology and is considered a symptom of the disease. Compounds that attenuate stress-induced hyperthermia in animals are believed to be useful in treating anxiety disorders in humans. Generalized anxiety disorder and post-traumatic stress disorder are among the disorders that can be treated with these compounds. A common minimally invasive method for analyzing stress-induced hyperthermia is by measuring body temperature and stress-induced body temperature increase through a rectal thermometer. Male Fisher F-344 rats weighing 275-350 g (Haaran, Indianapolis, IN) were tested. All animals were individually housed with freely available food and automated water and maintained on a 12 hour light / dark cycle (lit at 06:00). The animals were fasted for approximately 12-18 hours prior to the experiment and the experiment was performed during the light phase. (IP) to rats at a dose volume of 1 mL / kg with 0.3, 1, 3 and 10 mg / kg of compounds of the invention. The vehicle achieved a pH of 5-7 by the addition of brine + NaOH. The mGluR5 antagonist MTEP (3 - [(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine with strong antianxiety-like activity demonstrated in preclinical models was used as a comparator (5 mg / Kg, IP route, dissolved in water). Immediately after administration, the rat was returned to his home cage, and the experimenter turned off the lamp and left the room. The dosing chamber was dark for one hour for the remainder of the pretreatment period.

After the pretreatment period, the reference body temperature was determined by inserting the rats separately into the adjoining room illuminated brightly and inserting a rectal probe lubricated with mineral oil. Body temperature was evaluated using a Fiji Temp BAT-12® microprobe thermometer (Physitemp Instruments Inc., Clifton, NJ) with a PHYSITEMP RET-2® rat rectal probe. To measure deep body temperature, the probe was inserted approximately 2 cm into the rectum (this is the reference body temperature (T1) (Celsius temperature)). After 10 minutes, secondary body temperature measurements were recorded (T2). The difference in body temperature (T2 - T1) was defined as stress - induced hyperthermia. The compounds of the present invention as compared to vehicle response stress were defined the dose causing a 35% reduction in the high temperature causes the reaction to T ₃₅ capacity.

Example 2 yielded a T ₃₅ of 0.57 mg / kg as measured in this assay as practiced above. Example 5 was measured in this assay as practiced above, yielding a T ₃₅ of 6.4 mg / kg. These results demonstrate that the compounds within the scope of the present invention are useful drugs for anxiety disorders. More particularly, compounds within the scope of the present invention may be useful drugs for generalized anxiety disorder and / or post-traumatic stress disorder.

In vitro PepT1 GlySar inhibition screen and IC ₅₀ determinations

The PepT1 assay was set up to investigate the ability of the prodrug to interact with the enteroviral transporter PepT1.

HeLa cells derived from human intestine (American Type Culture Collection) were inoculated with 10% fetal bovine serum (FBS), 0.1 mM nonessential amino acid (NEAA) and 100 units / mL penicillin + 100 μg / mL streptomycin (Invitrogen, Cat # SH30243) in a 5% CO ₂ humidified atmosphere at 37 &lt; 0 &gt; C. Cell lines were used for sub-40 passages and were then discarded. Frozen cells in a 1 ml vial were thawed for 1-2 minutes in a water bath and added to 5 mL of cell culture at 37 ° C. Each T-flask was provided with 8.5 mL of fresh medium and 1.5 mL of cell stock. Cells were passaged twice for one week. This is done by rinsing the flask with 10 mL of phosphate buffered saline-ethylenediaminetetraacetic acid (PBS-EDTA), separating the cells by adding 2 mL of trypsin for 2-5 minutes, adding 8 mL of fresh medium, Lt; / RTI &gt; To obtain a 1: 6 cell dilution, each new flask was provided with a combination of 8.5 mL of fresh medium and 1.5 mL of cell stock. Cells were incubated at &lt; RTI ID = 0.0 &gt; 37 C &lt; / RTI &gt;

70-80% of the full-grown cells in the T-flask were plated one day before the transfection procedure. The flask containing the cell stock was treated with PBS-EDTA and trypsin to separate the cells and the transfection medium was used from this point on. Transfection media consisted of Dulbecco's modified Eagle's medium (DMEM) + NEAA. To each well was added 0.5 mL of cell mixture (1.3 x ¹⁰ &lt; ⁵ &gt; is the desired cell concentration) and the cells were incubated overnight at 37 &lt; 0 &gt; C. Cells were transfected with PEPT1 24 hours prior to challenge. The transfection mixture was generated by mixing 600 μL of serum-free transfection medium, 18 μL of FuGene 6 (Roche Diagnostics) and 11 μg of PepT1 DNA. The transfection reagent-DNA complex was incubated for 20 minutes and 24 μL of reagent-DNA complex was added to each well.

Inhibition of PEPT1-mediated [glycyl-1-2- ¹⁴ C] glycylsarcosine (GlySar) uptake activity was measured in cells cultured in 24-hour post transfection in 24-well plates as previously disclosed (Zhang et al. 2004. J. Pharm. Exper. Ther. 310: 437-445). To measure the ability of compounds of the invention to inhibit the absorption of [ ¹⁴ C] Gly-Sar, the prodrug was incubated with 5 μM [ ¹⁴ C] Gly-Sar (Moravek Biochemicals) and 20 μM cold Gly -Sar with PepTl transiently transfected HeLa cells grown at 80-90% at 5 mM in pH 6.0 adsorption medium. Absorbent medium consisted of 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl 2, 0.8 mM MgSO 4, 5 mM glucose, 25 mM tris (hydroxymethyl) aminomethane buffer (tris (TRIS)). The solution was then made pH 6.0 using 2- (N-morpholino) ethanesulfonic acid. Incubation volume was 500 [mu] L, and was performed at room temperature for 3 minutes. To stop the absorption at the end of the incubation time, the absorption medium was aspirated from the cell monolayer and 500 μL of ice-cold PBS was added to the wells. Cells were washed three times with 500 μL of room temperature PBS without Ca ^{+ 2} and Mg ^{+ 2} . Then, to dissolve the cells in 1% Triton (Triton) X100 H ₂ O solution of 300 μL. 200 [mu] L aliquots were taken and the radioactivity was determined by liquid scintillation counts to determine [ ¹⁴ C] Gly-Sar present in each incubation well. Control groups without inhibitors were set up and the percent inhibition of each prodrug was calculated for the control group. A negative control (glycine) and two positive controls (cephadoxil and cephalexin) were performed in parallel with each experiment to verify the validity of the assay system. Prodrugs with GlySar uptake inhibition equal to or better than cephalexin were considered acceptable. The mean ± SD was 10.1 ± 9.5% (n = 19) for glycine, 53.2 ± 13.2% (n = 19) for cephadoxil and 37.5 ± 14.7% (n = 18) for cephalexin.

In the case of the PepT IC ₅₀ assay, the prodrug was incubated in the concentration range (0.0625 to 25 mM) in the presence of 5 μM [ ¹⁴ C] Gly-Sar and 20 μM cold Gly-Sar. The incubation and sampling procedure was exactly the same as the PepTl screen described above. ^[14 C] Evaluation for the Gly-Sar uptake data for each prodrug concentration, which was calculated the IC ₅₀ value.

Examples 5, 6 and 7 obtained inhibition of hPepTl [3H] Gly-Sar uptake by 89%, 87% and 78% at 5 mM, respectively, as measured in this assay as practiced above. Examples 5 and 6 obtained hPepTl [3H] Gly-Sar absorption inhibition IC50 of 1.98 mM and 0.25 mM, respectively, as measured in this assay as practiced above. These results demonstrate that compounds within the scope of the present invention are orally absorbed through the PepTl transporter.

In vitro intestinal tract drug hydrolysis assay

The frozen human duodenum homogenate (1: 2 tissue: buffer ratio using 100 mM trisphosphate buffer, pH 7.4) was obtained from Celsius In Vitro Technologies (Baltimore, MD) But did not contain both fluoride (PMSF) and EDTA.

Each lot of the human duodenum was obtained from a single donor, scraped into the bowel, and the sections were frozen separately. All initial tissue collection was performed at 4 ° C and immediately frozen at -70 ° C. Human intestinal homogenates were thawed and incubated immediately after dilution to a final protein concentration of 0.5 mg / mL in 100 mM PBS buffer, pH 7.4.

Incubations were performed in 96-well plates and all prodrugs were run in duplicate on a daily basis. A stock progenitor drug solution was prepared at a concentration of 1 mM in water. A 200 μL aliquot of 0.5 mg / mL intestinal homogenate and 196 μL of 100 mM PBS buffer were placed in a 96-well plate in a 37 ° C water bath. Using a 96-well pipettor, 4 μL of 1 mM prodrug solution was transferred into the homogenate. Immediately after addition of the prodrug (time 0) and 1 hour incubation, an automated single-dose 96-well pipettor was used to remove 50 μL samples of the incubation mixture and 200 μL of 100 ng / Methanol was added directly to the quenching solution. The samples were then centrifuged at 3500 rpm for 5 minutes at &lt; RTI ID = 0.0 &gt; 10 C. &lt; / RTI &gt; The supernatant (200 [mu] L) was transferred to a final 96 well PCR plate and sealed for analysis by LC / MS / MS.

The concentration of the hydrolyzed active metabolite in the incubation mixture was measured on a Sciex API 4000 tetrahedron mass spectrometer with analyst version 1.4.2, TurboIonSpray, positive ionization and selective reaction monitoring (SRM) LC / MS / MS detection. A Waters Atlantis® T3 (20 x 2.1 mm, 5 μM) HPLC column was used at a flow rate of 1.0 mL / min at ambient temperature and with a mobile phase gradient of 0.1% mobile phase A → 99% mobile phase A. Mobile phase A was 1000: 5 water: heptafluorobuteric acid and mobile phase B was 1: 1 methanol: glacial acetic acid.

The concentration of the hydrolyzed active metabolite in the long incubation mixture was determined from the standard curve generated by repeating 2-fold dilutions starting at 10 [mu] M in 100 mM PBS pH 7.4 and subsequently quenched with the same methanol- Respectively. The mean and standard deviation were calculated using Microsoft® Office Excel® 2007. The amount of hydrolysis was determined as the molar percentage of the active metabolite formed relative to the added prodrug concentration. The hydrolysis of the positive control internal prodrug A to the internal active metabolite Drug A was performed with an average of 75.3% (n = 20) per batch. The final values were then normalized for the formation of the internal active metabolite Drug A &lt; RTI ID = 0.0 &gt;

Examples 5, 6 and 7 show that 36% (n = 3, SD = 2.7) and 44% (n = 3, SD = 4.1) ) And 34% (n = 1) of human intestinal hydrolysis. These results demonstrate that the compounds within the scope of the present invention are hydrolyzed in the human field.

In vitro human liver S-9 homogenate hydrolysis assay

Liver S9 fraction was obtained from Xenotech LLC (Renexa, MO). Lot was from a pool of two donors, one male and one female. Liver S9 fraction was prepared and diluted using homogenization buffer consisting of 50 mM Tris, pH 7.4 and 150 mM potassium chloride at 4 [deg.] C without EDTA. After incubation of the prodrug in hepatic homogenate at 37 占 폚 for 2 hours, the concentration of active metabolite was determined by LC / MS / MS. Hydrolysis of clopidogrel to clopidogrel carboxylic acid was used as an assay positive control.

Incubations were performed in 96-well format and all prodrugs were run in duplicate on a daily basis. A stock progenitor drug solution was prepared at a concentration of 1 mM in water. The human liver S9 fraction was diluted to a final protein concentration of 0.5 mg / ml in 100 mM PBS buffer, pH 7.4.

A 200 μL aliquot of 0.5 mg / mL human liver S-9 homogenate and 196 μL of 100 mM PBS buffer were placed in a 96-well plate in a 37 ° C water bath. 4 uL of 1 mM prodrug solution was transferred into the homogenate using a 96-well pipettor. To ensure that the hydrolysis was not due to chemical instability, the prodrug was also incubated alone with PBS buffer without liver S-9. Immediately after addition of the prodrug (time 0) and 1 hour incubation, a 50 μL sample of the incubation mixture was removed using an automated disposable simultaneous 96-well pipettor, and 200 μL containing 100 ng / mL internal standard Of methanol quenching solution. The sample was then centrifuged at 3500 rpm for 5 minutes at &lt; RTI ID = 0.0 &gt; 10 C. &lt; / RTI &gt; The supernatant (200 uL) was transferred to a final 96 well PCR plate and sealed for analysis by LC / MS / MS.

LC / MS / MS quantification of the active metabolites formed during incubation was performed on the Siex API 4000 (Analyst version 1.4.2, Turboon spray, positive ionization and selective reaction monitoring (SRM)). The HPLC column used was Waters Atlantis T3 (20 x 2.1 mm, 5 m) at a mobile flow rate of 1.0 mL / min at ambient temperature. Mobile phase A was 1000: 5 water: heptafluorobutylic acid and mobile phase B was 1: 1 methanol: glacial acetic acid. A mobile phase gradient beginning at 99.9 / 0.1 mobile phase ratio A / B and ending at 1/99 was used.

The concentration of the hydrolyzed active metabolite in the incubation mixture was determined from a standard curve generated by repeating 2-fold dilutions starting at 10 μM in 100 mM PBS pH 7.4 and subsequently quenched with the same methanol-internal standard solution as the sample. The mean and standard deviation were calculated using Microsoft® Office Excel® 2007. The final value is expressed as the molar percentage of the active metabolite formed relative to the concentration of the prodrug added. Hydrolysis of clopidogrel to clopidogrel carboxylic acid was used as a positive control and the mean was 73.0% (n = 27).

Examples 8 and 9 obtained 23% and 59.3% of human S9 hydrolysis, respectively, as measured in this assay as practiced above. These results demonstrate that the compounds within the scope of the present invention are hydrolyzed in the human liver.

The data demonstrate that the exemplified amino acid prodrug inhibits the absorption of the PepTl substrate GlySar as better or better than cephadoxyl and cephalexin (Zhang et al., 2004. JPET 310: 437-445) Suggesting the possibility of human oral absorption via the PepT1 transporter. In addition to prodrug absorption, prodrug hydrolysis is essential to obtain active metabolites when introduced into the body. The in vitro hydrolysis studies of the present invention suggested that the exemplified amino acid prodrugs could be hydrolyzed by the human intestine. The hydrolysis of the exemplified diester prodrug occurred in human liver homogenate, suggesting that the exemplified diester prodrug could be hydrolyzed in humans following oral exposure. These data indicate the possibility that the amino acid prodrugs exemplified together and the exemplified diester prodrugs are hydrolyzed in humans to release active metabolites.

Pharmacokinetic assay

Fast transfected male Sprague Dawley rats were dosed intravenously with 1 mg / kg of Example 2 or 7.5 mg / kg of Example 5, respectively, in a standard crossover design (N = 3, rats receiving both intravenous and oral doses, respectively) kg &lt; / RTI &gt; (equivalent to 5 mg / kg molar equivalents of Example 2) by oral gavage. For intravenous administration, Example 2 was dissolved in water and Example 5 was diluted in water with the addition of hydroxyethylcellulose (1%), polysorbate 80 (0.25%) and defoamer 1510-US (0.05%) Aqueous vehicle. The cannula was surgically implanted to facilitate a series of blood collections. Blood was collected in EDTA tubes over a period of 0-24 hours after administration, centrifuged, and the plasma was stored frozen until analysis.

Plasma samples were thawed, 50 μL aliquots were transferred to 96-well plates, 50 μL of internal standard solution was added, and the samples were mixed. Subsequently, 300 μL of acetonitrile was added, the sample was vortexed for 3 minutes, and centrifuged. A 300 μL aliquot of the supernatant was transferred to a separate plate and evaporated under nitrogen at 40 ° C. The residue was reconstituted in 100 [mu] L of 0.5% heptafluorobutyric acid in water, vortexed and centrifuged. 20 [mu] L aliquots were subsequently analyzed by LC / MS / MS using a Shimadzu autosampler and HPLC system intercon- nected with AB X 4000 mass spectrometer under a positive ion turbo spray mode. The MS / MS transfer was 283.2? 44.2 amu for prodrug 5 and 212.1? 103.1 amu for active 2. (B) acetonitrile-water (1: 1, v / v) on a moving bed of acetonitrile, Atlantis T3, 50 x 2.1 mm, 1.0 mL / min and (A) 0.2% formic acid in water Column, and a gradient of 2% B to 98% B over 0.8 minutes was used. The residence time of Example 2 was approximately 0.53 minutes.

Pharmacokinetic parameters were calculated from plasma concentration data using Watson for Windows (Thermo Scientific). Relative oral bioavailability was determined by comparing the area under the plasma concentration time curve (AUC) of the active Example 2 after intravenous administration with the AUC of the active metabolite after oral administration of the prodrug Example 5.

Successful prodrugs must be well absorbed after oral administration and subsequently hydrolyzed to release the active metabolite into the systemic circulation. Relative bioavailability included both parameters by comparing the AUC of the active metabolite after oral administration of the prodrug with the AUC of the activity after intravenous administration of the active compound. In male rats, the relative bioavailability of the active metabolite after oral administration of prodrug Example 5, as measured in this assay as practiced as described above, was 60 + 14% (mean + -standard deviation) Demonstrating that Example 5 was well absorbed and hydrolyzed extensively to produce in vivo active metabolites.

Claims (27)

Claims 1. Compounds of the general formula < RTI ID = 0.0 > (I) &lt;
(I)

In this formula,
R ¹ is hydrogen, R ² is hydrogen, and R ³ is hydrogen;
R ¹ is hydrogen, R ² is (2S) -2-aminopropanoyl, and R ³ is hydrogen;
R ¹ is hydrogen, R ² is (2S) -2-amino-4-methylsulfanyl-butanoyl and R ³ is hydrogen;
R ¹ is hydrogen, R ² is 2-aminoacetyl, and R ³ is hydrogen;
R ¹ is benzyl, R ² is hydrogen and R ³ is benzyl; or
R ¹ is (2-fluorophenyl) methyl, R ² is hydrogen, and R ³ is (2-fluorophenyl) methyl. The compound according to claim 1, which is (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid, A pharmaceutically acceptable salt. 2. The method according to claim 1, wherein the compound is (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylic acid hydrochloride compound. 2. The compound according to claim 1, which is (1S, 2S, 5R, 6S) -2 - [[(2S) -2-aminopropanoyl] amino] spiro [bicyclo [3.1.0] hexane- Propane] -2,6-dicarboxylic acid or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, which is (1S, 2S, 5R, 6S) -2 - [[(2S) -2-aminopropanoyl] amino] spiro [bicyclo [3.1.0] hexane- Propane] -2, 6-dicarboxylic acid hydrochloride. 2. The compound according to claim 1, which is (1S, 2S, 5R, 6S) -2 - [[(2S) -2-aminopropanoyl] amino] spiro [bicyclo [3.1.0] hexane- Propane] -2,6-dicarboxylic acid dihydrate. 2. The compound according to claim 1, which is (1S, 2S, 5R, 6S) -2 - [[(2S) -2-aminopropanoyl] amino] spiro [bicyclo [3.1.0] hexane- Propane] -2,6-dicarboxylic acid; 1,4-dioxane (1: 0.5); Lt; / RTI &gt; 4. The compound of claim 1 which is (1S, 2S, 5R, 6S) -2 - [[(2S) -2-amino- 4,1'-cyclopropane] -2,6-dicarboxylic acid, or a pharmaceutically acceptable salt thereof. 4. The compound of claim 1 which is (1S, 2S, 5R, 6S) -2 - [[(2S) -2-amino- 4,1'-cyclopropane] -2,6-dicarboxylic acid hydrochloride. 3. The compound according to claim 1, which is (1S, 2S, 5R, 6S) -2 - [(2-aminoacetyl) amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -Dicarboxylic acid or a pharmaceutically acceptable salt thereof. 3. The compound according to claim 1, which is (1S, 2S, 5R, 6S) -2 - [(2-aminoacetyl) amino] spiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] - dicarboxylic acid hydrochloride. The method of claim 1, wherein the dibenzyl (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate Or a pharmaceutically acceptable salt thereof. The process of claim 1, wherein the dibenzyl (1S, 2S, 5R, 6S) -2-aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] -2,6-dicarboxylate; 1,4-dioxane; Lt; / RTI &gt; 2. The compound according to claim 1, which is bis [(2-fluorophenyl) methyl] (1S, 2S, 5R, 6S) -2- aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] 2,6-dicarboxylate, or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, which is bis [(2-fluorophenyl) methyl] (1S, 2S, 5R, 6S) -2- aminospiro [bicyclo [3.1.0] hexane-4,1'-cyclopropane] 2,6-dicarboxylate hydrochloride. 16. A pharmaceutical composition comprising a compound or salt of any one of claims 1 to 15 and a pharmaceutically acceptable carrier, diluent or excipient. 16. A compound or salt according to any one of claims 1 to 15 for use in therapy. 16. A compound or salt according to any one of claims 1 to 15 for use in the treatment of a neurological disorder selected from the group consisting of persistent pain, neuropathic pain, chronic inflammatory pain and visceral pain. 19. The compound or salt of claim 18, wherein the nervous system disorder is persistent pain. 19. The compound or salt of claim 18, wherein the neurological disorder is neuropathic pain. 19. A compound or salt according to claim 18, wherein the nervous system disorder is chronic inflammatory pain. 19. A compound or salt according to claim 18, wherein the nervous system disorder is a visceral pain. 16. A compound or salt according to any one of claims 1 to 15 for use in the treatment of a psychotic disorder selected from the group consisting of schizophrenia, bipolar disorder, generalized anxiety disorder and post-traumatic stress disorder. 24. The compound or salt of claim 23, wherein the mental disorder is schizophrenia. 24. The compound or salt of claim 23, wherein the mental disorder is a bipolar disorder. 24. The compound or salt of claim 23, wherein the mental disorder is generalized anxiety disorder. 24. The compound or salt of claim 23, wherein the mental disorder is post-traumatic stress disorder.